Treatment of B-cell malignancies by a combination JAK and PI3K inhibitors

ABSTRACT

This invention relates to methods of treating B-cell malignancies using a combination of inhibitors of JAK1 and/or JAK2 and inhibitors of PI3Kδ.

This application is a continuation of U.S. Ser. No. 14/680,659, filedApr. 7, 2015, claims the benefit of priority of 61/976,815, filed Apr.8, 2014, which is incorporated herein by reference in its entirety.

TECHNICAL FIELD

This invention relates to methods of treating B-cell malignancies usinga combination of inhibitors of JAK1 and/or JAK2 and inhibitors of PI3Kδ.

BACKGROUND

The B-cell receptor (BCR) is present on both normal and most malignantB-cells. Engagement of the BCR provides important survival signals, andinterruption of the BCR signal can lead to B-cell death. Studiesperformed with siRNA to inhibit BCR expression have shown thatconstitutive signaling by the BCR is critical for the survival andproliferation of human B-cell lymphomas. The primary role of BCRsignaling in these cells appears to be activation of spleen tyrosinekinase (Syk) which in turn leads to several downstream events thatpromote cell survival, including activation of Bruton tyrosine kinase(BTK), phosphatidylinositol 3 kinase (PI3K), and AKT. A number of B-cellmalignancies, including diffuse large B-cell lymphomas (DLBCL), havebeen shown to be particularly dependent upon BCR survival signals asevidenced by their sensitivity to genetic and pharmacological inhibitionof BCR signaling components in vitro. It has been shown that DLBCL cellsengage PI3K, augmenting anti-apoptotic NF-kB signaling and survivalsignals and that inhibition of the PI3K/AKT pathway synergizes withNF-kB inhibition in killing DLBCL cell lines in vitro.

Aberrant activation of JAKs, through production of cytokines and growthfactors, has also been associated with increased malignant cellproliferation and survival in a number of tumor types. JAKs activate anumber of downstream pathways implicated in the proliferation andsurvival of malignant cells including the STATs, a family of importantlatent transcription factors. Of clinical relevance, levels of serumIL-10 and IL-6, which signal through the JAKs, have been found to beelevated in patients with DLBCL compared to normal controls (Gupta etal, 2012). Further, patients with high serum IL-10 levels were shown tohave a shorter event-free survival (Gupta et al, 2012). Within the JAKfamily of kinases, JAK1 has been shown to cooperate with JAK2, JAK3, andTYK2 and to play a dominant role in mediating the signaling of a numberof inflammatory cytokines including IL-6, IL-10 and interferon.

In DLBCL, JAK pathway activation occurs through both autocrine andparacrine mechanisms. In the tumor cells, BCR signaling leads toincreased IL-6 and IL-10 production through activation of the NF-kBpathway (Lam et al, 2008). A subset of DLBCLs has been characterized ashaving high expression of STAT3, IL-6, and/or IL-10 and it has beenshown that JAK inhibition is cytotoxic in these DLBCL cell lines andsynergizes with NF-kB inhibitors. In addition to JAK/STAT pathwayactivation through autocrine pathways, the stromal compartment can alsoprovide a source of these cytokines in a paracrine manner (Hodge et al,2005).

For these reasons, there is a need to develop new therapies that can beused to treat B-cell malignancies, such as DLBCL. This invention isdirected to this need and others.

DESCRIPTION OF THE DRAWINGS

FIG. 1A depicts Western blot analysis probing for IL6 and IL10 forvarious DLBCL cell lines.

FIG. 1B depicts Western blot analysis for actin and p-Stat3 for Pfeiffercells treated with IL6 or IL10.

FIG. 2A depicts % inhibition in the cellular proliferation assay inPfeiffer cells as a function of the concentration of Compound 28 withvehicle (DMSO), DMSO+IL10, and DMSO+IL10+ruxolitinib.

FIG. 2B depicts % inhibition in the cellular proliferation assay inPfeiffer cells as a function of the concentration of Compound 28 withvehicle (DMSO), DMSO+IL10, and DMSO+IL10+Compound 7.

FIG. 3 depicts % inhibition in the cellular proliferation assay in HBL-1cells as a function of the concentration of Compound 28 with vehicle(DMSO), DMSO+IL10, and DMSO+IL10+ruxolitinib.

FIG. 4 depicts Western blot analysis of Pfeiffer cells after treatmentwith vehicle (DMSO), ruxolitinib, Compound 28, or Compound 28 andruxolitinib with or without IL10.

FIG. 5 depicts Western blot analysis of Pfeiffer cells after treatmentwith vehicle (DMSO), Compound 7, Compound 28, or Compound 28 andCompound 7 with or without IL10.

FIG. 6 depicts % inhibition in the cellular proliferation assay inPfeiffer cells as a function of the concentration of Compound 28 withvehicle (DMSO), DMSO+IL10, and DMSO+IL10+Compound 16.

FIG. 7 depicts Annexin-V staining of Pfeiffer cells treated withCompound 28+/−Compound 16, showing a synergistic apoptosis inductionfrom the combination therapy.

FIG. 8 depicts Western blot analysis of Pfeiffer cells after treatmentwith Compound 28+/−Compound 16 showing effect on STAT3 and pAKT.

SUMMARY

The present application provides a method of treating a B-cellmalignancy in a patient in need thereof, comprising administering tosaid patient: (a) an inhibitor of JAK1 and/or JAK2; and (b) an inhibitorof PI3Kδ.

The present application further provides a method of treating a diseaseselected from diffuse large B-cell lymphoma, chronic lymphocyticleukemia (CLL), Non-Hodgkin lymphoma, hairy cell leukemia, Mantle celllymphoma, small lymphocytic lymphoma, follicular lymphoma,lymphoplasmacytic lymphoma, extranodal marginal zone lymphoma, Hodgkin'slymphoma, Burkitt lymphoma, Waldenstrom's macroglobulinemia,prolymphocytic leukemia, acute lymphoblastic leukemia, myelofibrosis,mucosa-associated lymphatic tissue (MALT) lymphoma, mediastinal (thymic)large B-cell lymphoma, lymphomatoid granulomatosis, splenic marginalzone lymphoma, primary effusion lymphoma, intravascular large B-celllymphoma, plasma cell leukemia, extramedullary plasmacytoma, smoulderingmyeloma (aka asymptomatic myeloma), monoclonal gammopathy ofundetermined significance (MGUS), activated B-cell like (ABC) diffuselarge B cell lymphoma (ABC-DLBCL), and germinal center B cell (GCB)diffuse large B cell lymphoma (GCB-DLBCL) in a patient in need thereof,comprising administering to said patient: (a) an inhibitor of JAK1and/or JAK2; and (b) an inhibitor of PI3Kδ.

In some embodiments of the methods, the inhibitor of JAK1 and/or JAK2 isselected from:

-   3-cyclopentyl-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]propanenitrile;-   3-[1-(6-chloropyridin-2-yl)pyrrolidin-3-yl]-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]propanenitrile;-   3-(1-[1,3]oxazolo[5,4-b]pyridin-2-ylpyrrolidin-3-yl)-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]propanenitrile;-   4-[(4-{3-cyano-2-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]propyl}piperazin-1-yl)carbonyl]-3-fluorobenzonitrile;-   4-[(4-{3-cyano-2-[3-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrrol-1-yl]propyl}piperazin-1-yl)carbonyl]-3-fluorobenzonitrile;-   {1-{1-[3-Fluoro-2-(trifluoromethyl)isonicotinoyl]piperidin-4-yl}-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-3-yl}acetonitrile;-   4-{3-(cyanomethyl)-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-1-yl}-N-[4-fluoro-2-(trifluoromethyl)phenyl]piperidine-1-carboxamide;-   [3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]-1-(1-{[2-(trifluoromethyl)pyrimidin-4-yl]carbonyl}piperidin-4-yl)azetidin-3-yl]acetonitrile;-   [trans-1-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]-3-(4-{[2-(trifluoromethyl)pyrimidin-4-yl]carbonyl}piperazin-1-yl)cyclobutyl]acetonitrile;-   {trans-3-(4-{[4-[(3-hydroxyazetidin-1-yl)methyl]-6-(trifluoromethyl)pyridin-2-yl]oxy}piperidin-1-yl)-1-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]cyclobutyl}acetonitrile;-   {trans-3-(4-{[4-{[(2S)-2-(hydroxymethyl)pyrrolidin-1-yl]methyl}-6-(trifluoromethyl)pyridin-2-yl]oxy}piperidin-1-yl)-1-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]cyclobutyl}acetonitrile;-   {trans-3-(4-{[4-{[(2R)-2-(hydroxymethyl)pyrrolidin-1-yl]methyl}-6-(trifluoromethyl)pyridin-2-yl]oxy}piperidin-1-yl)-1-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]cyclobutyl}acetonitrile;-   4-(4-{3-[(dimethylamino)methyl]-5-fluorophenoxy}piperidin-1-yl)-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]butanenitrile;-   5-{3-(cyanomethyl)-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-1-yl}-N-isopropylpyrazine-2-carboxamide;-   4-{3-(cyanomethyl)-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-1-yl}-2,5-difluoro-N-[(1S)-2,2,2-trifluoro-1-methylethyl]benzamide;-   5-{3-(cyanomethyl)-3-[4-(1H-pyrrolo[2,3-b]pyridin-4-yl)-1H-pyrazol-1-yl]azetidin-1-yl}-N-isopropylpyrazine-2-carboxamide;-   {1-(cis-4-{[6-(2-hydroxyethyl)-2-(trifluoromethyl)pyrimidin-4-yl]oxy}cyclohexyl)-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-3-yl}acetonitrile;-   {1-(cis-4-{[4-[(ethylamino)methyl]-6-(trifluoromethyl)pyridin-2-yl]oxy}cyclohexyl)-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-3-yl}acetonitrile;-   {1-(cis-4-{[4-(1-hydroxy-1-methylethyl)-6-(trifluoromethyl)pyridin-2-yl]oxy}cyclohexyl)-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-3-yl}acetonitrile;-   {1-(cis-4-{[4-{[(3R)-3-hydroxypyrrolidin-1-yl]methyl}-6-(trifluoromethyl)pyridin-2-yl]oxy}cyclohexyl)-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-3-yl}acetonitrile;-   {1-(cis-4-{[4-{[(3S)-3-hydroxypyrrolidin-1-yl]methyl}-6-(trifluoromethyl)pyridin-2-yl]oxy}cyclohexyl)-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-3-yl}acetonitrile;-   {trans-3-(4-{[4-({[(1S)-2-hydroxy-1-methylethyl]amino}methyl)-6-(trifluoromethyl)pyridin-2-yl]oxy}piperidin-1-yl)-1-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]cyclobutyl}acetonitrile;-   {trans-3-(4-{[4-({[(2R)-2-hydroxypropyl]amino}methyl)-6-(trifluoromethyl)pyridin-2-yl]oxy}piperidin-1-yl)-1-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]cyclobutyl}acetonitrile;-   {trans-3-(4-{[4-({[(2S)-2-hydroxypropyl]amino}methyl)-6-(trifluoromethyl)pyridin-2-yl]oxy}piperidin-1-yl)-1-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]cyclobutyl}acetonitrile;-   {trans-3-(4-{[4-(2-hydroxyethyl)-6-(trifluoromethyl)pyridin-2-yl]oxy}piperidin-1-yl)-1-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]cyclobutyl}acetonitrile;-   ((2R,5S)-5-{2-[(1R)-1-hydroxyethyl]-1H-imidazo[4,5-d]thieno[3,2-b]pyridin-1-yl}tetrahydro-2H-pyran-2-yl)acetonitrile;-   4-[3-(cyanomethyl)-3-(3′,5′-dimethyl-1H,1′H-4,4′-bipyrazol-1-yl)azetidin-1-yl]-2,5-difluoro-N-[(1S)-2,2,2-trifluoro-1-methylethyl]benzamide;

and pharmaceutically acceptable salts of any of the aforementioned.

In some embodiments of the methods, the inhibitor of PI3Kδ is selectedfrom:

-   7-(1-(9H-purin-6-ylamino)ethyl)-6-(3-fluorophenyl)-3-methyl-5H-thiazolo[3,2-a]pyrimidin-5-one;-   (S)-7-(1-(9H-purin-6-ylamino)ethyl)-6-(3-fluorophenyl)-3-methyl-5H-thiazolo[3,2-a]pyrimidin-5-one;-   4-[1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-6-chloro-2-{1-[(2S)-2-hydroxypropyl]azetidin-3-yl}-3-methoxybenzonitrile;-   4-[1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-6-chloro-2-[1-(2-hydroxyethyl)azetidin-3-yl]-3-methoxybenzonitrile;-   5-{3-[1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-6-cyano-2-ethoxy-5-methylphenyl}-N,N-dimethylpyridine-2-carboxamide;-   4-{3-[1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-5-chloro-2-ethoxy-6-fluorophenyl}pyrrolidin-2-one;    and-   N-{1-[5-chloro-8-(3-fluorophenyl)cinnolin-7-yl]ethyl}-9H-purin-6-amine;-   4-chloro-3′-fluoro-3-methyl-6-[1-(9H-purin-6-ylamino)ethyl]biphenyl-2-carbonitrile;

and pharmaceutically acceptable salts of any of the aforementioned.

The present application also provides an inhibitor of JAK1 and/or JAK2for use in combination with PI3Kδ inhibitor for the treatment of aB-cell malignancy or any of the diseases embodied herein.

The present application further provides use of an inhibitor of JAK1and/or JAK2 and an PI3Kδ inhibitor for preparation of a medicament fortreatment of a B-cell malignancy or any of the diseases embodied herein.

DETAILED DESCRIPTION

The present application provides, inter alia, a method of treating adisease selected from diffuse large B-cell lymphoma, chronic lymphocyticleukemia (CLL), Non-Hodgkin lymphoma, hairy cell leukemia, Mantle celllymphoma, small lymphocytic lymphoma, follicular lymphoma,lymphoplasmacytic lymphoma, extranodal marginal zone lymphoma, Hodgkin'slymphoma, Burkitt lymphoma, Waldenstrom's macroglobulinemia,prolymphocytic leukemia, acute lymphoblastic leukemia, myelofibrosis,mucosa-associated lymphatic tissue (MALT) lymphoma, mediastinal (thymic)large B-cell lymphoma, lymphomatoid granulomatosis, splenic marginalzone lymphoma, primary effusion lymphoma, intravascular large B-celllymphoma, plasma cell leukemia, extramedullary plasmacytoma, smoulderingmyeloma (aka asymptomatic myeloma), monoclonal gammopathy ofundetermined significance (MGUS), activated B-cell like (ABC) diffuselarge B cell lymphoma (ABC-DLBCL), and germinal center B cell (GCB)diffuse large B cell lymphoma (GCB-DLBCL) in a patient in need thereof,comprising administering to said patient: (a) an inhibitor of JAK1and/or JAK2; and (b) an inhibitor of PI3Kδ.

In some embodiments, the non-Hodgkin lymphoma is non-Hodgkin lymphoma(NHL) is relapsed or refractory NHL or recucurrent follicular NHL.

In some embodiments, the disease is diffuse large B cell lymphoma(DLBCL).

In some embodiments, the disease is activated B-cell like (ABC) diffuselarge B cell lymphoma (ABC-DLBCL) or germinal center B cell (GCB)diffuse large B cell lymphoma (GCB-DLBCL).

In some embodiments, the inhibitor of JAK1 and/or JAK2 and the inhibitorof PI3Kδ are administered simultaneously.

In some embodiments, the inhibitor of JAK1 and/or JAK2 and the inhibitorof PI3Kδ are administered sequentially.

In some embodiments, the inhibitor of JAK1 and/or JAK2 is selective forJAK1 and JAK1 over JAK3 and TYK2. In some embodiments, the inhibitor ofJAK1 and/or JAK2 is selective for JAK1 over JAK2, JAK3, and TYK2. Forexample, some of the compounds described herein, or a pharmaceuticallyacceptable salt thereof, preferentially inhibit JAK1 over one or more ofJAK2, JAK3, and TYK2. In some embodiments, the compounds inhibit JAK1preferentially over JAK2 (e.g., have a JAK1/JAK2 IC₅₀ ratio>1). In someembodiments, the compounds or salts are about 10-fold more selective forJAK1 over JAK2. In some embodiments, the compounds or salts are about3-fold, about 5-fold, about 10-fold, about 15-fold, or about 20-foldmore selective for JAK1 over JAK2 as calculated by measuring IC₅₀ at 1mM ATP (e.g., see Example A).

In some embodiments, the inhibitor of JAK1 and/or JAK2 is3-cyclopentyl-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]propanenitrile.In some embodiments, the inhibitor of JAK1 and/or JAK2 is(3R)-3-cyclopentyl-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]propanenitrile(ruxolitinib; also known as INCB018424). Ruxolitinib has an IC₅₀ of lessthan 10 nM at 1 mM ATP (assay A) at JAK1 and JAK2.3-Cyclopentyl-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]propanenitrileand ruxolitinib can be made by the procedure described in U.S. Pat. No.7,598,257 (Example 67), filed Dec. 12, 2006, which is incorporatedherein by reference in its entirety. In some embodiments, the inhibitorof JAK1 and/or JAK2 is(3R)-3-cyclopentyl-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]propanenitrilephosphoric acid salt.

In some embodiments, the inhibitor of JAK1 and/or JAK2 is a compound ofTable 1, or a pharmaceutically acceptable salt thereof. The compounds inTable 1 are selective JAK1 inhibitors (selective over JAK2, JAK3, andTYK2). The IC₅₀s obtained by the method of Assay A at 1 mM ATP are shownin Table 1.

TABLE 1 JAK1 IC₅₀ JAK2/ # Prep. Name Structure (nM) JAK1  1 Example 1herein ((2R,5S)-5-{2-[(1R)-1- hydroxyethyl]-1H- imidazo[4,5-d]thieno[3,2-b]pyridin- 1-yl}tetrahydro-2H- pyran-2-yl)acetonitrile

++ >10  2 Example 2 herein 4-[3-(cyanomethyl)-3- (3′,5′-dimethyl-1H,1′H-4,4′-bipyrazol-1- yl)azetidin-1-yl]-2,5- difluoro-N-[(1S)-2,2,2-trifluoro-1- methylethyl]benzamide

+++ >10  3 US 2010/ 0298334 (Example 2)^(a) 3-[1-(6-chloropyridin-2-yl)pyrrolidin-3-yl]-3-[4- (7H-pyrrolo[2,3- d]pyrimidin-4-yl)-1H-pyrazol-1- yl]propanenitrile

+ >10  4 US 2010/ 0298334 (Example 13c) 3-(1-[1,3]oxazolo[5,4-b]pyridin-2-ylpyrrolidin- 3-yl)-3-[4-(7H- pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1- yl]propanenitrile

+ >10  5 US 2011/ 0059951 (Example 12) 4-[(4-{3-cyano-2-[4-(7H-pyrrolo[2,3- d]pyrimidin-4-yl)-1H- pyrazol-1- yl]propyl}piperazin-1-yl)carbonyl]-3- fluorobenzonitrile

+ >10  6 US 2011/ 0059951 (Example 13) 4-[(4-{3-cyano-2-[3-(7H-pyrrolo[2,3- d]pyrimidin-4-yl)-1H- pyrrol-1- yl]propyl}piperazin-1-yl)carbonyl]-3- fluorobenzonitrile

+ >10  7 US 2011/ 0224190 (Example 1) {1-{1-[3-Fluoro-2-(trifluoromethyl) isonicotinoyl] piperidin-4-yl}-3- [4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H- pyrazol-1-yl]azetidin-3- yl}acetonitrile

+ >10  8 US 2011/ 0224190 (Example 154) 4-{3-(Cyanomethyl)-3-[4-(7H-pyrrolo[2,3- d]pyrimidin-4-yl)-1H- pyrazol-1-yl]azetidin-1-yl}-N-[4-fluoro-2- (trifluoromethyl)phenyl] piperidine-1- carboxamide

+ >10  9 US 2011/ 0224190 (Example 85) [3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H- pyrazol-1-yl]-1-(1-{[2- (trifluoromethyl)pyrimidin-4- yl]carbonyl}piperidin-4- yl)azetidin-3- yl]acetonitrile

+ >10 10 US 2012/ 0149681 (Example 7b) [trans-1-[4-(7H-pyrrolo[2,3-d]pyrimidin- 4-yl)-1H-pyrazol-1-yl]- 3-(4-{[2-(trifluoromethyl) pyrimidin-4- yl]carbonyl}piperazin-1-yl)cyclobutyl]acetonitrilc

+ >10 11 US 2012/ 0149681 (Example 157) {trans-3-(4-{[4-[(3-hydroxyazetidin-1- yl)methyl]-6- (trifluoromethyl)pyridin-2-yl]oxy}piperidin-1- yl)-1-[4-(7H- pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1- yl]cyclobutyl}acetonitrile

+ >10 12 US 2012/ 0149681 (Example 161) {trans-3-(4-{[4-{[(2S)-2-(hydroxymethyl) pyrrolidin- 1-yl]methyl}-6- (trifluoromethyl)pyridin-2-yl]oxy}piperidin-1- yl)-1-[4-(7H- pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1- yl]cyclobutyl}acetonitrile

+ >10 13 US 2012/ 0149681 (Example 162) {trans-3-(4-{[4-{[(2R)-2-(hydroxymethyl) pyrrolidin- 1-yl]methyl}-6- (trifluoromethyl)pyridin-2-yl]oxy}piperidin-1- yl)-1-[4-(7H- pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1- yl]cyclobutyl}acetonitrile

+ >10 14 US 2012/ 0149682 (Example 20)^(b) 4-(4-{3- [(dimethylamino)methyl]-5- fluorophenoxy}piperidin- 1-yl)-3-[4-(7H-pyrrolo[2,3-d]pyrimidin- 4-yl)-1H-pyrazol-1- yl]butanenitrile

+ >10 15 US 2013/ 0018034 (Example 18) 5-{3-(cyanomethyl)-3-[4-(7H-pyrrolo[2,3- d]pyrimidin-4-yl)-1H- pyrazol-1-yl]azetidin-1-yl}-N- isopropylpyrazine-2- carboxamide

+ >10 16 US 2013/ 0018034 (Example 28) 4-{3-(cyanomethyl)-3-[4-(7H-pyrrolo[2,3- d]pyrimidin-4-yl)-1H- pyrazol-1-yl]azetidin-1-yl}-2,5-difluoro-N- [(1S)-2,2,2-trifluoro-1- methylethyl]benzamide

+ >10 17 US 2013/ 0018034 (Example 34) 5-{3-(cyanomethyl)-3-[4-(1H-pyrrolo[2,3- b|pyridin-4-yl)-1H- pyrazol-1-yl]azetidin-1- yl}-N-isopropylpyrazine-2- carboxamide

+ >10 18 US 2013/ 0045963 (Example 45) {1-(cis-4-{[6-(2-hydroxyethyl)-2- (trifluoromethyl) pyrimidin-4- yl]oxy}cyclohexyl)-3-[4-(7H-pyrrolo[2,3- d]pyrimidin-4-yl)-1H- pyrazol-1-yl]azetidin-3-yl}acetonitrile

+ >10 19 US 2013/ 0045963 (Example 65) {1-(cis-4-{[4-[(ethylamino)methyl]-6- (trifluoromethyl)pyridin-2-yl]oxy}cyclohexyl)-3- [4-(7H-pyrrolo[2,3- d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-3- yl}acetonitrile

+ >10 20 US 2013/ 0045963 (Example 69) {1-(cis-4-{[4-(1-hydroxy-1-methylethyl)- 6- (trifluoromethyl)pyridin-2-yl]oxy}cyclohexyl)-3- [4-(7H-pyrrolo[2,3- d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-3- yl}acetonitrile

+ >10 21 US 2013/ 0045963 (Example 95) {1-(cis-4-{[4-{[(3R)-3-hydroxypyrrolidin-1- yl]methyl}-6- (trifluoromethyl)pyridin-2-yl]oxy}cyclohexyl)-3- [4-(7H-pyrrolo[2,3- d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-3- yl}acetonitrile

+ >10 22 US 2013/ 0045963 (Example 95) {1-(cis-4-{[4-{[(3S)-3-hydroxypyrrolidin-1- yl]methyl}-6- (trifluoromethyl)pyridin-2-yl]oxy}cyclohexyl)-3- [4-(7H-pyrrolo[2,3- d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-3- yl}acetonitrile

+ >10 23 US 2014/ 0005166 (Example 1) {trans-3-(4-{[4-({[(1S)-2-hydroxy-1- methylethyl]amino} methyl)-6- (trifluoromethyl)pyridin-2-yl]oxy}piperidin-1- yl)-1-[4-(7H- pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1- yl]cyclobutyl}acetonitrile

+ >10 24 US 2014/ 0005166 (Example 14) {trans-3-(4-{[4-({[(2R)- 2-hydroxypropyl]amino} methyl)-6- (trifluoromethyl)pyridin-2-yl]oxy}piperidin-1- yl)-1-[4-(7H- pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1- yl]cyclobutyl}acetonitrile

+ >10 25 US 2014/ 0005166 (Example 15) {trans-3-(4-{[4-({[(2S)- 2-hydroxypropyl]amino} methyl)-6- (trifluoromethyl)pyridin-2-yl]oxy}piperidin-1- yl)-1-[4-(7H- pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1- yl]cyclobutyl}acetonitrile

+ >10 26 US 2014/ 0005166 (Example 20) {trans-3-(4-{[4-(2-hydroxyethyl)-6- (trifluoromethyl)pyridin- 2-yl]oxy}piperidin-1-yl)-1-[4-(7H- pyrrolo[2,3-d]pyrimidin- 4-yl)-1H-pyrazol-1-yl]cyclobutyl}acetonitrile

+ >10 + means <10 nM (see Example A for assay conditions) ++ means ≤100nM (see Example A for assay conditions) +++ means ≤300 nM (see Example Afor assay conditions) ^(a)Data for enantiomer 1 ^(b)Data for enantiomer2

In some embodiments, the inhibitor of JAK1 and/or JAK2 is{1-{1-[3-fluoro-2-(trifluoromethyl)isonicotinoyl]piperidin-4-yl}-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-3-yl}acetonitrile,or a pharmaceutically acceptable salt thereof.

In some embodiments, the inhibitor of JAK1 and/or JAK2 is{1-{1-[3-fluoro-2-(trifluoromethyl)isonicotinoyl]piperidin-4-yl}-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-3-yl}acetonitrileadipic acid salt.

In some embodiments, the inhibitor of JAK1 and/or JAK2 is4-{3-(cyanomethyl)-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-1-yl}-2,5-difluoro-N-[(1S)-2,2,2-trifluoro-1-methylethyl]benzamide,or a pharmaceutically acceptable salt thereof.

In some embodiments, the inhibitor of JAK1 and/or JAK2 is selected from(R)-3-[1-(6-chloropyridin-2-yl)pyrrolidin-3-yl]-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]propanenitrile,(R)-3-(1-[1,3]oxazolo[5,4-b]pyridin-2-ylpyrrolidin-3-yl)-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]propanenitrile,(R)-4-[(4-{3-cyano-2-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]propyl}piperazin-1-yl)carbonyl]-3-fluorobenzonitrile,(R)-4-[(4-{3-cyano-2-[3-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrrol-1-yl]propyl}piperazin-1-yl)carbonyl]-3-fluorobenzonitrile,or(R)-4-(4-{3-[(dimethylamino)methyl]-5-fluorophenoxy}piperidin-1-yl)-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]butanenitrile,(S)-3-[1-(6-chloropyridin-2-yl)pyrrolidin-3-yl]-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]propanenitrile,(S)-3-(1-[1,3]oxazolo[5,4-b]pyridin-2-ylpyrrolidin-3-yl)-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]propanenitrile,(S)-4-[(4-{3-cyano-2-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]propyl}piperazin-1-yl)carbonyl]-3-fluorobenzonitrile,(S)-4-[(4-{3-cyano-2-[3-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrrol-1-yl]propyl}piperazin-1-yl)carbonyl]-3-fluorobenzonitrile,(S)-4-(4-{3-[(dimethylamino)methyl]-5-fluorophenoxy}piperidin-1-yl)-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]butanenitrile;and pharmaceutically acceptable salts of any of the aforementioned.

In some embodiments, the compounds of Table 1 are prepared by thesynthetic procedures described in US Patent Publ. No. 2010/0298334,filed May 21, 2010, US Patent Publ. No. 2011/0059951, filed Aug. 31,2010, US Patent Publ. No. 2011/0224190, filed Mar. 9, 2011, US PatentPubl. No. 2012/0149681, filed Nov. 18, 2011, US Patent Publ. No.2012/0149682, filed Nov. 18, 2011, US Patent Publ. 2013/0018034, filedJun. 19, 2012, US Patent Publ. No. 2013/0045963, filed Aug. 17, 2012,and US Patent Publ. No. 2014/0005166, filed May 17, 2013, each of whichis incorporated herein by reference in its entirety.

In some embodiments, the inhibitor of JAK1 and/or JAK2 is selected fromthe compounds of US Patent Publ. No. 2010/0298334, filed May 21, 2010,US Patent Publ. No. 2011/0059951, filed Aug. 31, 2010, US Patent Publ.No. 2011/0224190, filed Mar. 9, 2011, US Patent Publ. No. 2012/0149681,filed Nov. 18, 2011, US Patent Publ. No. 2012/0149682, filed Nov. 18,2011, US Patent Publ. 2013/0018034, filed Jun. 19, 2012, US Patent Publ.No. 2013/0045963, filed Aug. 17, 2012, and US Patent Publ. No.2014/0005166, filed May 17, 2013, each of which is incorporated hereinby reference in its entirety.

The inhibitors of PI3Kδ described herein can be selective. By“selective” is meant that the compound binds to or inhibits a kinasewith greater affinity or potency, respectively, compared to at least oneother kinase. In some embodiments, the compounds described herein areselective inhibitors of PI3Kδ (e.g., over PI3Kα, PI3Kβ and PI3Kγ). Insome embodiments, selectivity can be at least about 2-fold, 5-fold,10-fold, at least about 20-fold, at least about 50-fold, at least about100-fold, at least about 200-fold, at least about 500-fold or at leastabout 1000-fold. Selectivity can be measured by methods routine in theart. In some embodiments, selectivity can be tested at the K_(m) ATPconcentration of each enzyme. In some embodiments, the selectivity ofcompounds described herein can be determined by cellular assaysassociated with particular PI3K kinase activity.

In some embodiments, the inhibitor of PI3Kδ is a compound shown in Table2. The compounds of Table 2 have been tested in Assay B and shown to beinhibitors of PI3Kδ with the IC₅₀s in Table 2.

TABLE 2 PI3Kδ IC₅₀ # Prep. Name Structure (nM) 27 US 2011/0015212(Example 10) 7-(1-(9H-purin-6- ylamino)ethyl)-6-(3-fluorophenyl)-3-methyl- 5H-thiazolo[3,2- a]pyrimidin-5-one

+ 28 US 2011/0015212 (Example 15) (S)-7-(1-(9H-purin-6-ylamino)ethyl)-6-(3- fluorophenyl)-3-methyl- 5H-thiazolo[3,2-a]pyrimidin-5-one

+ 29 US 2013/ 0059835 (Example 269) 4-[1-(4-amino-3-methyl-1H-pyrazolo[3,4- d]pyrimidin-1-yl)ethyl]- 6-chloro-2-{1-[(2S)-2-hydroxypropyl]azetidin- 3-yl}-3- methoxybenzonitrile

+ 30 US 2013/ 0059835 (Example 268) 4-[1-(4-amino-3-methyl-1H-pyrazolo[3,4- d]pyrimidin-1-yl)ethyl]- 6-chloro-2-[1-(2-hydroxyethyl)azetidin-3- yl]-3- methoxybenzonitrile

+ 31 US 2013/ 0059835 (Example 314) 5-{3-[1-(4-amino-3-methyl-1H-pyrazolo[3,4- d]pyrimidin-1-yl)ethyl]- 6-cyano-2-ethoxy-5-methylphenyl}-N,N- dimethylpyridine-2- carboxamide

+ 32a, 32b, 32c, 32d US 2013/ 0059835 (Example 345- 348 (fourdiastereomers)) Compound 32a, 32b, 32c, and 3 2d are Examples 345, 346,347, and 348 respectively 4-{3-[1-(4-amino-3- methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]- 5-chloro-2-ethoxy-6- fluorophenyl}pyrrolidin-2-one

32a (++), 32b (+)   32c (+)   32d (++)  33 US 2011/0183985 (Example 17-single enantiomer) N-{1-[5-chloro-8-(3- fluorophenyl)cinnolin-7-yl]ethyl}-9H-purin-6- amine

+ 34 US 2012/ 0157430 4-chloro-3′-fluoro-3- methyl-6-[1-(9H-purin-6-ylamino)ethyl]biphenyl- 2-carbonitrile

+++ + means <50 nM ++ means 50 nM to 200 nM +++ means 50 nM to 100 nM

In some embodiments, the inhibitor of PI3Kδ is selected from:

-   (S)-4-(3-((S)-1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-5-chloro-2-ethoxy-6-fluorophenyl)pyrrolidin-2-one;-   (R)-4-(3-((S)-1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-5-chloro-2-ethoxy-6-fluorophenyl)pyrrolidin-2-one;-   (S)-4-(3-((R)-1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-5-chloro-2-ethoxy-6-fluorophenyl)pyrrolidin-2-one;-   (R)-4-(3-((R)-1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-5-chloro-2-ethoxy-6-fluorophenyl)pyrrolidin-2-one;-   N-{(1S)-1-[5-chloro-8-(3-fluorophenyl)cinnolin-7-yl]ethyl}-9H-purin-6-amine;

and pharmaceutically acceptable salts of any of the aforementioned.

In some embodiments, the inhibitor of PI3Kδ is(S)-7-(1-(9H-purin-6-ylamino)ethyl)-6-(3-fluorophenyl)-3-methyl-5H-thiazolo[3,2-a]pyrimidin-5-one,or a pharmaceutically acceptable salt thereof.

In some embodiments, the inhibitor of PI3Kδ is4-[1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-6-chloro-2-{1-[(2S)-2-hydroxypropyl]azetidin-3-yl}-3-methoxybenzonitrile,or a pharmaceutically acceptable salt thereof

In some embodiments, the inhibitor of PI3Kδ is4-[1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-6-chloro-2-[1-(2-hydroxyethyl)azetidin-3-yl]-3-methoxybenzonitrile,or a pharmaceutically acceptable salt thereof

In some embodiments, the inhibitor of PI3Kδ is5-{3-[1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-6-cyano-2-ethoxy-5-methylphenyl}-N,N-dimethylpyridine-2-carboxamide,or a pharmaceutically acceptable salt thereof

In some embodiments, the inhibitor of PI3Kδ is selected from:

-   4-[(R)-1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-6-chloro-2-{1-[(2S)-2-hydroxypropyl]azetidin-3-yl}-3-methoxybenzonitrile;-   4-[1(R)-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-6-chloro-2-[1-(2-hydroxyethyl)azetidin-3-yl]-3-methoxybenzonitrile;-   5-{3-[1(R)-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-6-cyano-2-ethoxy-5-methylphenyl}-N,N-dimethylpyridine-2-carboxamide;-   4-[(S)-1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-6-chloro-2-{1-[(2S)-2-hydroxypropyl]azetidin-3-yl}-3-methoxybenzonitrile;-   4-[1(S)-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-6-chloro-2-[1-(2-hydroxyethyl)azetidin-3-yl]-3-methoxybenzonitrile;-   5-{3-[1(S)-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-6-cyano-2-ethoxy-5-methylphenyl}-N,N-dimethylpyridine-2-carboxamide;

and pharmaceutically acceptable salts of any of the aforementioned.

In some embodiments, the inhibitor of PI3Kδ is a compound of US PatentPubl. No. US 2011/0015212, filed Jun. 28, 2010, US Patent Publ. No.2013/0059835, filed Aug. 31, 2013, US Patent Publ. No. 2011/0183985,filed Dec. 17, 2010, or US Patent Publ. No. 2012/0157430, filed Dec. 19,2011, each of which is incorporated herein by reference in its entirety.

In some embodiments, the compounds of Table 2 are prepared by themethods in US Patent Publ. No. US 2011/0015212, filed Jun. 28, 2010, USPatent Publ. No. 2013/0059835, filed Aug. 31, 2013, US Patent Publ. No.2011/0183985, filed Dec. 17, 2010, or US Patent Publ. No. 2012/0157430,filed Dec. 19, 2011, each of which is incorporated herein by referencein its entirety.

In some embodiments, the inhibitor of JAK1 and/or JAK2 is(3R)-3-cyclopentyl-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]propanenitrile,or a pharmaceutically acceptable salt thereof; and(7-(1-(9H-purin-6-ylamino)ethyl)-6-(3-fluorophenyl)-3-methyl-5H-thiazolo[3,2-a]pyrimidin-5-one,or a pharmaceutically acceptable salt thereof.

In some embodiments, the inhibitor of JAK1 and/or JAK2 is{1-{1-[3-fluoro-2-(trifluoromethyl)isonicotinoyl]piperidin-4-yl}-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-3-yl}acetonitrile,or pharmaceutically acceptable salt thereof; and the inhibitor of PI3Kδis7-(1-(9H-purin-6-ylamino)ethyl)-6-(3-fluorophenyl)-3-methyl-5H-thiazolo[3,2-a]pyrimidin-5-one,or a pharmaceutically acceptable salt thereof.

In some embodiments, the inhibitor of JAK1 and/or JAK2 is4-{3-(cyanomethyl)-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-1-yl}-2,5-difluoro-N-[(1S)-2,2,2-trifluoro-1-methylethyl]benzamide,or pharmaceutically acceptable salt thereof; and the inhibitor of PI3Kδis7-(1-(9H-purin-6-ylamino)ethyl)-6-(3-fluorophenyl)-3-methyl-5H-thiazolo[3,2-a]pyrimidin-5-one,or a pharmaceutically acceptable salt thereof.

In some embodiments, the present application provides a method oftreating diffuse large B-cell lymphoma in a patient in need thereof,comprising administering to said patient(3R)-3-cyclopentyl-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]propanenitrile,or a pharmaceutically acceptable salt thereof and the inhibitor of PI3Kδis(7-(1-(9H-purin-6-ylamino)ethyl)-6-(3-fluorophenyl)-3-methyl-5H-thiazolo[3,2-a]pyrimidin-5-one,or a pharmaceutically acceptable salt thereof.

In some embodiments, the present application provides a method oftreating diffuse large B-cell lymphoma in a patient in need thereof,comprising administering to said patient{1-{1-[3-fluoro-2-(trifluoromethyl)isonicotinoyl]piperidin-4-yl}-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-3-yl}acetonitrile,or pharmaceutically acceptable salt thereof; and7-(1-(9H-purin-6-ylamino)ethyl)-6-(3-fluorophenyl)-3-methyl-5H-thiazolo[3,2-a]pyrimidin-5-one,or a pharmaceutically acceptable salt thereof.

In some embodiments, the present application provides a method oftreating diffuse large B-cell lymphoma in a patient in need thereof,comprising administering to said patient4-{3-(cyanomethyl)-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-1-yl}-2,5-difluoro-N-[(1S)-2,2,2-trifluoro-1-methylethyl]benzamide,or pharmaceutically acceptable salt thereof; and7-(1-(9H-purin-6-ylamino)ethyl)-6-(3-fluorophenyl)-3-methyl-5H-thiazolo[3,2-a]pyrimidin-5-one,or a pharmaceutically acceptable salt thereof.

The compounds described herein can be asymmetric (e.g., having one ormore stereocenters). All stereoisomers, such as enantiomers anddiastereomers, are intended unless otherwise indicated. Compounds thatcontain asymmetrically substituted carbon atoms can be isolated inoptically active or racemic forms. Methods on how to prepare opticallyactive forms from optically inactive starting materials are known in theart, such as by resolution of racemic mixtures or by stereoselectivesynthesis. Many geometric isomers of olefins, C═N double bonds, and thelike can also be present in the compounds described herein, and all suchstable isomers are contemplated in the present invention. Cis and transgeometric isomers of the compounds of the present invention aredescribed and may be isolated as a mixture of isomers or as separatedisomeric forms. In some embodiments, the compound has the(R)-configuration. In some embodiments, the compound has the(S)-configuration.

Resolution of racemic mixtures of compounds can be carried out by any ofnumerous methods known in the art. An example method includes fractionalrecrystallizaion using a chiral resolving acid which is an opticallyactive, salt-forming organic acid. Suitable resolving agents forfractional recrystallization methods are, for example, optically activeacids, such as the D and L forms of tartaric acid, diacetyltartaricacid, dibenzoyltartaric acid, mandelic acid, malic acid, lactic acid orthe various optically active camphorsulfonic acids such asβ-camphorsulfonic acid. Other resolving agents suitable for fractionalcrystallization methods include stereoisomerically pure forms ofα-methylbenzylamine (e.g., S and R forms, or diastereomerically pureforms), 2-phenylglycinol, norephedrine, ephedrine, N-methylephedrine,cyclohexylethylamine, 1,2-diaminocyclohexane, and the like.

Resolution of racemic mixtures can also be carried out by elution on acolumn packed with an optically active resolving agent (e.g.,dinitrobenzoylphenylglycine). Suitable elution solvent composition canbe determined by one skilled in the art.

Compounds described herein also include tautomeric forms. Tautomericforms result from the swapping of a single bond with an adjacent doublebond together with the concomitant migration of a proton. Tautomericforms include prototropic tautomers which are isomeric protonationstates having the same empirical formula and total charge. Exampleprototropic tautomers include ketone—enol pairs, amide—imidic acidpairs, lactam—lactim pairs, enamine—imine pairs, and annular forms wherea proton can occupy two or more positions of a heterocyclic system, forexample, 1H- and 3H-imidazole, 1H-, 2H- and 4H-1,2,4-triazole, 1H- and2H-isoindole, and 1H- and 2H-pyrazole. Tautomeric forms can be inequilibrium or sterically locked into one form by appropriatesubstitution.

Compounds described herein can also include all isotopes of atomsoccurring in the intermediates or final compounds. Isotopes includethose atoms having the same atomic number but different mass numbers.For example, isotopes of hydrogen include tritium and deuterium.

The term, “compound,” as used herein is meant to include allstereoisomers, geometric iosomers, tautomers, and isotopes of thestructures depicted. Compounds herein identified by name or structure asone particular tautomeric form are intended to include other tautomericforms unless otherwise specified.

All compounds, and pharmaceutically acceptable salts thereof, can befound together with other substances such as water and solvents (e.g.hydrates and solvates) or can be isolated.

In some embodiments, the compounds described herein, or salts thereof,are substantially isolated. By “substantially isolated” is meant thatthe compound is at least partially or substantially separated from theenvironment in which it was formed or detected. Partial separation caninclude, for example, a composition enriched in the compounds describedherein. Substantial separation can include compositions containing atleast about 50%, at least about 60%, at least about 70%, at least about80%, at least about 90%, at least about 95%, at least about 97%, or atleast about 99% by weight of the compounds described herein, or saltthereof. Methods for isolating compounds and their salts are routine inthe art.

The phrase “pharmaceutically acceptable” is employed herein to refer tothose compounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of human beings and animals without excessive toxicity,irritation, allergic response, or other problem or complication,commensurate with a reasonable benefit/risk ratio.

The expressions, “ambient temperature” and “room temperature” or “rt” asused herein, are understood in the art, and refer generally to atemperature, e.g. a reaction temperature, that is about the temperatureof the room in which the reaction is carried out, for example, atemperature from about 20° C. to about 30° C.

The present invention also includes pharmaceutically acceptable salts ofthe compounds described herein. As used herein, “pharmaceuticallyacceptable salts” refers to derivatives of the disclosed compoundswherein the parent compound is modified by converting an existing acidor base moiety to its salt form. Examples of pharmaceutically acceptablesalts include, but are not limited to, mineral or organic acid salts ofbasic residues such as amines; alkali or organic salts of acidicresidues such as carboxylic acids; and the like. The pharmaceuticallyacceptable salts of the present invention include the conventionalnon-toxic salts of the parent compound formed, for example, fromnon-toxic inorganic or organic acids. The pharmaceutically acceptablesalts of the present invention can be synthesized from the parentcompound which contains a basic or acidic moiety by conventionalchemical methods. Generally, such salts can be prepared by reacting thefree acid or base forms of these compounds with a stoichiometric amountof the appropriate base or acid in water or in an organic solvent, or ina mixture of the two; generally, non-aqueous media like ether, ethylacetate, alcohols (e.g., methanol, ethanol, iso-propanol, or butanol) oracetonitrile (ACN) are preferred. Lists of suitable salts are found inRemington's Pharmaceutical Sciences, 17th ed., Mack Publishing Company,Easton, Pa., 1985, p. 1418 and Journal of Pharmaceutical Science, 66, 2(1977), each of which is incorporated herein by reference in itsentirety.

As used herein, the term “individual” or “patient,” usedinterchangeably, refers to any animal, including mammals, preferablymice, rats, other rodents, rabbits, dogs, cats, swine, cattle, sheep,horses, or primates, and most preferably humans.

In some embodiments, the inhibitors are administered in atherapeutically effective amount. As used herein, the phrase“therapeutically effective amount” refers to the amount of activecompound or pharmaceutical agent that elicits the biological ormedicinal response that is being sought in a tissue, system, animal,individual or human by a researcher, veterinarian, medical doctor orother clinician. In some embodiments, the dosage of the compound, or apharmaceutically acceptable salt thereof, administered to a patient orindividual is about 1 mg to about 2 g, about 1 mg to about 1000 mg,about 1 mg to about 500 mg, about 1 mg to about 200 mg, about 1 mg toabout 100 mg, about 1 mg to 50 mg, or about 50 mg to about 500 mg.

As used herein, the term “treating” or “treatment” refers to one or moreof (1) inhibiting the disease; for example, inhibiting a disease,condition or disorder in an individual who is experiencing or displayingthe pathology or symptomatology of the disease, condition or disorder(i.e., arresting further development of the pathology and/orsymptomatology); and (2) ameliorating the disease; for example,ameliorating a disease, condition or disorder in an individual who isexperiencing or displaying the pathology or symptomatology of thedisease, condition or disorder (i.e., reversing the pathology and/orsymptomatology) such as decreasing the severity of disease.

Combination Therapies

One or more additional pharmaceutical agents such as, for example,chemotherapeutics, anti-inflammatory agents, steroids,immunosuppressants, as well as Bcr-Abl, Flt-3, EGFR, HER2, c-MET, VEGFR,PDGFR, cKit, IGF-1R, RAF, FAK, Akt mTOR, PIM, and AKT (e.g., AKT1, AKT2,or AKT3) kinase inhibitors such as, for example, those described in WO2006/056399, or other agents such as, therapeutic antibodies can be usedin combination with the compounds of the present invention for treatmentof PI3K-associated diseases, disorders or conditions. The one or moreadditional pharmaceutical agents can be administered to a patientsimultaneously or sequentially.

Example antibodies for use in combination therapy include but are notlimited to Trastuzumab (e.g. anti-HER2), Ranibizumab (e.g. anti-VEGF-A),Bevacizumab (trade name Avastin, e.g. anti-VEGF, Panitumumab (e.g.anti-EGFR), Cetuximab (e.g. anti-EGFR), Rituxan (anti-CD20) andantibodies directed to c-MET.

One or more of the following agents may be used in combination with thecompounds of the present invention and are presented as a non limitinglist: a cytostatic agent, cisplatin, doxorubicin, taxotere, taxol,etoposide, irinotecan, camptostar, topotecan, paclitaxel, docetaxel,epothilones, tamoxifen, 5-fluorouracil, methoxtrexate, temozolomide,cyclophosphamide, SCH 66336, R115777, L778,123, BMS 214662, Iressa,Tarceva, antibodies to EGFR, Gleevec™, intron, ara-C, adriamycin,cytoxan, gemcitabine, Uracil mustard, Chlormethine, Ifosfamide,Melphalan, Chlorambucil, Pipobroman, Triethylenemelamine,Triethylenethiophosphoramine, Busulfan, Carmustine, Lomustine,Streptozocin, Dacarbazine, Floxuridine, Cytarabine, 6-Mercaptopurine,6-Thioguanine, Fludarabine phosphate, oxaliplatin, leucovirin,ELOXATIN™, Pentostatine, Vinblastine, Vincristine, Vindesine, Bleomycin,Dactinomycin, Daunorubicin, Doxorubicin, Epirubicin, Idarubicin,Mithramycin, Deoxycoformycin, Mitomycin-C, L-Asparaginase, Teniposide17.alpha.-Ethinylestradiol, Diethylstilbestrol, Testosterone,Prednisone, Fluoxymesterone, Dromostanolone propionate, Testolactone,Megestrolacetate, Methylprednisolone, Methyltestosterone, Prednisolone,Triamcinolone, Chlorotrianisene, Hydroxyprogesterone, Aminoglutethimide,Estramustine, Medroxyprogesteroneacetate, Leuprolide, Flutamide,Toremifene, goserelin, Cisplatin, Carboplatin, Hydroxyurea, Amsacrine,Procarbazine, Mitotane, Mitoxantrone, Levamisole, Navelbene,Anastrazole, Letrazole, Capecitabine, Reloxafine, Droloxafine,Hexamethylmelamine, Avastin, herceptin, Bexxar, Velcade, Zevalin,Trisenox, Xeloda, Vinorelbine, Porfimer, Erbitux, Liposomal, Thiotepa,Altretamine, Melphalan, Trastuzumab, Lerozole, Fulvestrant, Exemestane,Fulvestrant, Ifosfomide, Rituximab, C225, Campath, Clofarabine,cladribine, aphidicolon, rituxan, sunitinib, dasatinib, tezacitabine,Smll, fludarabine, pentostatin, triapine, didox, trimidox, amidox, 3-AP,MDL-101,731, bendamustine (Treanda), ofatumumab, or GS-1101 (also knownas CAL-101).

Example chemotherapeutics include proteosome inhibitors (e.g.,bortezomib), thalidomide, revlimid, and DNA-damaging agents such asmelphalan, doxorubicin, cyclophosphamide, vincristine, etoposide,carmustine, and the like.

Example steroids include coriticosteroids such as dexamethasone orprednisone.

Example Bcr-Abl inhibitors include the compounds, and pharmaceuticallyacceptable salts thereof, of the genera and species disclosed in U.S.Pat. No. 5,521,184, WO 04/005281, and U.S. Ser. No. 60/578,491.

Example suitable Flt-3 inhibitors include compounds, and theirpharmaceutically acceptable salts, as disclosed in WO 03/037347, WO03/099771, and WO 04/046120.

Example suitable RAF inhibitors include compounds, and theirpharmaceutically acceptable salts, as disclosed in WO 00/09495 and WO05/028444.

Example suitable FAK inhibitors include compounds, and theirpharmaceutically acceptable salts, as disclosed in WO 04/080980, WO04/056786, WO 03/024967, WO 01/064655, WO 00/053595, and WO 01/014402.

Example suitable mTOR inhibitors include compounds, and theirpharmaceutically acceptable salts, as disclosed in WO 2011/025889.

In some embodiments, the compounds of the invention can be used incombination with one or more other kinase inhibitors including imatinib,particularly for treating patients resistant to imatinib or other kinaseinhibitors.

In some embodiments, the compounds of the invention can be used incombination with a chemotherapeutic in the treatment of cancer, such asmultiple myeloma, and may improve the treatment response as compared tothe response to the chemotherapeutic agent alone, without exacerbationof its toxic effects. Examples of additional pharmaceutical agents usedin the treatment of multiple myeloma, for example, can include, withoutlimitation, melphalan, melphalan plus prednisone [MP], doxorubicin,dexamethasone, and Velcade (bortezomib). Further additional agents usedin the treatment of multiple myeloma include Bcr-Abl, Flt-3, RAF and FAKkinase inhibitors. Additive or synergistic effects are desirableoutcomes of combining a PI3K inhibitor of the present invention with anadditional agent. Furthermore, resistance of multiple myeloma cells toagents such as dexamethasone may be reversible upon treatment with thePI3K inhibitor of the present invention. The agents can be combined withthe present compound in a single or continuous dosage form, or theagents can be administered simultaneously or sequentially as separatedosage forms.

In some embodiments, a corticosteroid such as dexamethasone isadministered to a patient in combination with the compounds of theinvention where the dexamethasone is administered intermittently asopposed to continuously.

In some further embodiments, combinations of the compounds of theinvention with other therapeutic agents can be administered to a patientprior to, during, and/or after a bone marrow transplant or stem celltransplant.

Pharmaceutical Formulations and Dosage Forms

When employed as pharmaceuticals, the compounds described herein can beadministered in the form of pharmaceutical compositions. Thesecompositions can be prepared in a manner well known in thepharmaceutical art, and can be administered by a variety of routes,depending upon whether local or systemic treatment is desired and uponthe area to be treated. Administration may be topical (includingtransdermal, epidermal, ophthalmic and to mucous membranes includingintranasal, vaginal and rectal delivery), pulmonary (e.g., by inhalationor insufflation of powders or aerosols, including by nebulizer;intratracheal or intranasal), oral or parenteral. Parenteraladministration includes intravenous, intraarterial, subcutaneous,intraperitoneal intramuscular or injection or infusion; or intracranial,e.g., intrathecal or intraventricular, administration. Parenteraladministration can be in the form of a single bolus dose, or may be, forexample, by a continuous perfusion pump. Pharmaceutical compositions andformulations for topical administration may include transdermal patches,ointments, lotions, creams, gels, drops, suppositories, sprays, liquidsand powders. Conventional pharmaceutical carriers, aqueous, powder oroily bases, thickeners and the like may be necessary or desirable. Thisinvention also includes pharmaceutical compositions which contain, asthe active ingredient, the compound described herein or apharmaceutically acceptable salt thereof, in combination with one ormore pharmaceutically acceptable carriers (excipients). In someembodiments, the composition is suitable for topical administration. Inmaking the compositions of the invention, the active ingredient istypically mixed with an excipient, diluted by an excipient or enclosedwithin such a carrier in the form of, for example, a capsule, sachet,paper, or other container. When the excipient serves as a diluent, itcan be a solid, semi-solid, or liquid material, which acts as a vehicle,carrier or medium for the active ingredient. Thus, the compositions canbe in the form of tablets, pills, powders, lozenges, sachets, cachets,elixirs, suspensions, emulsions, solutions, syrups, aerosols (as a solidor in a liquid medium), ointments containing, for example, up to 10% byweight of the active compound, soft and hard gelatin capsules,suppositories, sterile injectable solutions, and sterile packagedpowders.

In preparing a formulation, the active compound can be milled to providethe appropriate particle size prior to combining with the otheringredients. If the active compound is substantially insoluble, it canbe milled to a particle size of less than 200 mesh. If the activecompound is substantially water soluble, the particle size can beadjusted by milling to provide a substantially uniform distribution inthe formulation, e.g. about 40 mesh.

The compounds described herein may be milled using known millingprocedures such as wet milling to obtain a particle size appropriate fortablet formation and for other formulation types. Finely divided(nanoparticulate) preparations of the compounds described herein can beprepared by processes known in the art, e.g., see International App. No.WO 2002/000196.

Some examples of suitable excipients include lactose, dextrose, sucrose,sorbitol, mannitol, starches, gum acacia, calcium phosphate, alginates,tragacanth, gelatin, calcium silicate, microcrystalline cellulose,polyvinylpyrrolidone, cellulose, water, syrup, and methyl cellulose. Theformulations can additionally include: lubricating agents such as talc,magnesium stearate, and mineral oil; wetting agents; emulsifying andsuspending agents; preserving agents such as methyl- andpropylhydroxy-benzoates; sweetening agents; and flavoring agents. Thecompositions of the invention can be formulated so as to provide quick,sustained or delayed release of the active ingredient afteradministration to the patient by employing procedures known in the art.

The compositions can be formulated in a unit dosage form, each dosagecontaining from about 5 to about 1000 mg (1 g), more usually about 100to about 500 mg, of the active ingredient. The term “unit dosage forms”refers to physically discrete units suitable as unitary dosages forhuman subjects and other mammals, each unit containing a predeterminedquantity of active material calculated to produce the desiredtherapeutic effect, in association with a suitable pharmaceuticalexcipient.

In some embodiments, the compositions of the invention contain fromabout 5 to about 50 mg of the active ingredient. One having ordinaryskill in the art will appreciate that this embodies compositionscontaining about 5 to about 10, about 10 to about 15, about 15 to about20, about 20 to about 25, about 25 to about 30, about 30 to about 35,about 35 to about 40, about 40 to about 45, or about 45 to about 50 mgof the active ingredient.

In some embodiments, the compositions of the invention contain fromabout 50 to about 500 mg of the active ingredient. One having ordinaryskill in the art will appreciate that this embodies compositionscontaining about 50 to about 100, about 100 to about 150, about 150 toabout 200, about 200 to about 250, about 250 to about 300, about 350 toabout 400, or about 450 to about 500 mg of the active ingredient.

In some embodiments, the compositions of the invention contain fromabout 500 to about 1000 mg of the active ingredient. One having ordinaryskill in the art will appreciate that this embodies compositionscontaining about 500 to about 550, about 550 to about 600, about 600 toabout 650, about 650 to about 700, about 700 to about 750, about 750 toabout 800, about 800 to about 850, about 850 to about 900, about 900 toabout 950, or about 950 to about 1000 mg of the active ingredient.

Similar dosages may be used of the compounds described herein in themethods and uses of the invention.

The active compound can be effective over a wide dosage range and isgenerally administered in a pharmaceutically effective amount. It willbe understood, however, that the amount of the compound actuallyadministered will usually be determined by a physician, according to therelevant circumstances, including the condition to be treated, thechosen route of administration, the actual compound administered, theage, weight, and response of the individual patient, the severity of thepatient's symptoms, and the like.

For preparing solid compositions such as tablets, the principal activeingredient is mixed with a pharmaceutical excipient to form a solidpreformulation composition containing a homogeneous mixture of acompound of the present invention. When referring to thesepreformulation compositions as homogeneous, the active ingredient istypically dispersed evenly throughout the composition so that thecomposition can be readily subdivided into equally effective unit dosageforms such as tablets, pills and capsules. This solid preformulation isthen subdivided into unit dosage forms of the type described abovecontaining from, for example, about 0.1 to about 1000 mg of the activeingredient of the present invention.

The tablets or pills of the present invention can be coated or otherwisecompounded to provide a dosage form affording the advantage of prolongedaction. For example, the tablet or pill can comprise an inner dosage andan outer dosage component, the latter being in the form of an envelopeover the former. The two components can be separated by an enteric layerwhich serves to resist disintegration in the stomach and permit theinner component to pass intact into the duodenum or to be delayed inrelease. A variety of materials can be used for such enteric layers orcoatings, such materials including a number of polymeric acids andmixtures of polymeric acids with such materials as shellac, cetylalcohol, and cellulose acetate.

The liquid forms in which the compounds and compositions of the presentinvention can be incorporated for administration orally or by injectioninclude aqueous solutions, suitably flavored syrups, aqueous or oilsuspensions, and flavored emulsions with edible oils such as cottonseedoil, sesame oil, coconut oil, or peanut oil, as well as elixirs andsimilar pharmaceutical vehicles.

Compositions for inhalation or insufflation include solutions andsuspensions in pharmaceutically acceptable, aqueous or organic solvents,or mixtures thereof, and powders. The liquid or solid compositions maycontain suitable pharmaceutically acceptable excipients as describedsupra. In some embodiments, the compositions are administered by theoral or nasal respiratory route for local or systemic effect.Compositions can be nebulized by use of inert gases. Nebulized solutionsmay be breathed directly from the nebulizing device or the nebulizingdevice can be attached to a face mask, tent, or intermittent positivepressure breathing machine. Solution, suspension, or powder compositionscan be administered orally or nasally from devices which deliver theformulation in an appropriate manner.

Topical formulations can contain one or more conventional carriers. Insome embodiments, ointments can contain water and one or morehydrophobic carriers selected from, for example, liquid paraffin,polyoxyethylene alkyl ether, propylene glycol, white Vaseline, and thelike. Carrier compositions of creams can be based on water incombination with glycerol and one or more other components, e.g.glycerinemonostearate, PEG-glycerinemonostearate and cetylstearylalcohol. Gels can be formulated using isopropyl alcohol and water,suitably in combination with other components such as, for example,glycerol, hydroxyethyl cellulose, and the like. In some embodiments,topical formulations contain at least about 0.1, at least about 0.25, atleast about 0.5, at least about 1, at least about 2, or at least about 5wt % of the compound described herein. The topical formulations can besuitably packaged in tubes of, for example, 100 g which are optionallyassociated with instructions for the treatment of the select indication,e.g., psoriasis or other skin condition.

The amount of compound or composition administered to a patient willvary depending upon what is being administered, the purpose of theadministration, such as prophylaxis or therapy, the state of thepatient, the manner of administration, and the like. In therapeuticapplications, compositions can be administered to a patient alreadysuffering from a disease in an amount sufficient to cure or at leastpartially arrest the symptoms of the disease and its complications.Effective doses will depend on the disease condition being treated aswell as by the judgment of the attending clinician depending uponfactors such as the severity of the disease, the age, weight and generalcondition of the patient, and the like.

The compositions administered to a patient can be in the form ofpharmaceutical compositions described above. These compositions can besterilized by conventional sterilization techniques, or may be sterilefiltered. Aqueous solutions can be packaged for use as is, orlyophilized, the lyophilized preparation being combined with a sterileaqueous carrier prior to administration. The pH of the compoundpreparations typically will be between 3 and 11, more preferably from 5to 9 and most preferably from 7 to 8. It will be understood that use ofcertain of the foregoing excipients, carriers, or stabilizers willresult in the formation of pharmaceutical salts.

The therapeutic dosage of a compound of the present invention can varyaccording to, for example, the particular use for which the treatment ismade, the manner of administration of the compound, the health andcondition of the patient, and the judgment of the prescribing physician.The proportion or concentration of a compound described herein in apharmaceutical composition can vary depending upon a number of factorsincluding dosage, chemical characteristics (e.g., hydrophobicity), andthe route of administration. For example, the compounds described hereincan be provided in an aqueous physiological buffer solution containingabout 0.1 to about 10% w/v of the compound for parenteraladministration. Some typical dose ranges are from about 1 μg/kg to about1 g/kg of body weight per day. In some embodiments, the dose range isfrom about 0.01 mg/kg to about 100 mg/kg of body weight per day. Thedosage is likely to depend on such variables as the type and extent ofprogression of the disease or disorder, the overall health status of theparticular patient, the relative biological efficacy of the compoundselected, formulation of the excipient, and its route of administration.Effective doses can be extrapolated from dose-response curves derivedfrom in vitro or animal model test systems.

The compositions of the invention can further include one or moreadditional pharmaceutical agents such as a chemotherapeutic, steroid,anti-inflammatory compound, or immunosuppressant, examples of which arelisted herein.

Kits

The present invention also includes pharmaceutical kits useful, forexample, in the treatment or prevention of PI3K-associated diseases ordisorders, such as cancer, which include one or more containerscontaining a pharmaceutical composition comprising a therapeuticallyeffective amount of a compound described herein. Such kits can furtherinclude, if desired, one or more of various conventional pharmaceuticalkit components, such as, for example, containers with one or morepharmaceutically acceptable carriers, additional containers, etc., aswill be readily apparent to those skilled in the art. Instructions,either as inserts or as labels, indicating quantities of the componentsto be administered, guidelines for administration, and/or guidelines formixing the components, can also be included in the kit.

EXAMPLES Example 1.((2R,5S)-5-{2-[(1R)-1-Hydroxyethyl]-1H-imidazo[4,5-d]thieno[3,2-b]pyridin-1-yl}tetrahydro-2H-pyran-2-yl)acetonitrile

Step 1. tert-Butyl(4S)-2,2-dimethyl-4-vinyl-1,3-oxazolidine-3-carboxylate

To a suspension of methyl triphenylphosphonium bromide (5.63 g, 15.8mmol) in tetrahydrofuran (140 mL) was added 2.5 M n-butyllithium inhexane (7.35 mL, 18.4 mmol). The deep red solution was stirred at 0° C.for 1 h. Then a solution of tert-butyl(4R)-4-formyl-2,2-dimethyl-1,3-oxazolidine-3-carboxylate (from Aldrich,3.01 g, 13.1 mmol) in tetrahydrofuran (7.3 mL) was added drop wise at 0°C. The red solution was warmed to room temperature and stirred for 12 h.Hexanes was added to the reaction mixture in 4:1 (v/v) ratio. Thesuspension was filtered through Celite and the filtrate concentrated.The resultant residue was purified by flash chromatography (eluting with10% ethyl acetate in hexanes) to give the desired compound as colorlessoil (1.92 g, 64%).

Step 2. tert-Butyl [(1S)-1-(hydroxymethyl)prop-2-en-1-yl]carbamate

To a solution of tert-butyl(4S)-2,2-dimethyl-4-vinyl-1,3-oxazolidine-3-carboxylate (1.90 g, 8.36mmol) in methanol (83 mL) was added p-toluenesulfonic acid monohydrate(0.80 g, 4.2 mmol) at 0° C. The mixture was slowly warmed to roomtemperature overnight. The reaction mixture was diluted with saturatedNaHCO₃ solution, concentrated, and then diluted with ethyl acetate. Theorganic layer was washed with sat. NaHCO₃ (2×) and brine, dried overNa₂SO₄, filtered and concentrated to give the desired product ascolorless oil (1.187 g, 76%). ¹H NMR (400 MHz, CDCl₃) δ 5.81 (1H, m),5.25 (2H, m), 4.90 (1H, m), 4.25 (1H, br s), 3.67 (2H, m), 1.45 (9H, s)ppm.

Step 3. tert-Butyl[(1S)-1-({[1-(hydroxymethyl)prop-2-en-1-yl]oxy}methyl)prop-2-en-1-yl]carbamate

To a flask was charged with tert-butyl[(1S)-1-(hydroxymethyl)prop-2-en-1-yl]carbamate (0.401 g, 2.14 mmol),tris(dibenzylideneacetone)dipalladium(0) (59 mg, 0.064 mmol),N,N′-(1S,2S)-cyclohexane-1,2-diylbis[2-(diphenylphosphino)-1-naphthamide](150 mg, 0.19 mmol), and 4-dimethylaminopyridine (78 mg, 0.64 mmol). Thereaction mixture was purged with N₂ three times, and then methylenechloride (21.3 mL), and 1.0 M triethylborane in THF (130 μL, 0.13 mmol)was added sequentially. After stirring for 10 min, 2-vinyloxirane (0.150g, 2.14 mmol) was added and the resulting mixture was stirred overnight.The reaction was diluted with dichloromethane and sat. NaHCO₃ solution.The organic layer was separated and dried over Na₂SO₄, filtered andconcentrated. The crude residue was purified with flash chromatography(eluting with 0-50% ethyl acetate/hexanes) to give the desired product(0.271 g, 49%). ¹H NMR (300 MHz, CDCl₃) δ 5.85 (1H, m), 5.67 (1H, m),5.84˜5.17 (4H, m), 4.83 (1H, m), 4.30 (1H, br s), 3.83 (1H, m), 3.69(1H, dd, J=4.5 and 6.9 Hz), 3.54 (2H, m), 3.36 (1H, dd, J=4.5 and 6.9Hz), 1.45 (9H, s) ppm.

Step 4.2-({(2S)-2-[(tert-Butoxycarbonyl)amino]but-3-en-1-yl}oxy)but-3-en-1-ylacetate

To a mixture of tert-butyl[(1S)-1-({[1-(hydroxymethyl)prop-2-en-1-yl]oxy}methyl)prop-2-en-1-yl]carbamate(268 mg, 1.04 mmol) in methylene chloride (10 mL) was added withtriethylamine (435 μL, 3.12 mmol). The mixture was cooled to 0° C., andacetyl chloride (150 μL, 2.1 mmol) was added drop wise. The reaction wasstirred at room temperature for 2 h, then quenched with water. Theorganic layer was concentrated and the resultant residue purified onsilica gel (eluting with 20% ethyl acetate/hexanes) to give the desiredproduct (0.26 g, 85%). LCMS calculated for C₁₀H₁₈NO₃ (M−100+H)⁺:m/z=200.1; Found: 200.1.

Step 5.{(5S)-5-[(tert-Butoxycarbonyl)amino]-5,6-dihydro-2H-pyran-2-yl}methylacetate

To a 500 mL 2-neck round bottom flask,benzylidene(dichloro)(1,3-dimesitylimidazolidin-2-id-2-yl)(tricyclohexylphosphoranyl)ruthenium(38 mg, 0.044 mmol) was added. After purged with nitrogen for 3 times,dichloromethane (anhydrous, 8 mL) was added followed by2-({(2S)-2-[(tert-butoxycarbonyl)amino]but-3-en-1-yl}oxy)but-3-en-1-ylacetate (265 mg, 0.885 mmol). The reaction mixture was stirred at roomtemperature for 15 h. The mixture was concentrated in vacuo. The residuewas purified via flash chromatography (eluting with hexanes to 25% EtOAcin hexanes) to give the desired product as a brown oil (0.205 g, 85%).LCMS calculated for C₉H₁₄NO₅ (M+H-Bu+H)⁺: m/z=216.1; Found: 216.1. ¹HNMR (300 MHz, CDCl₃) δ 5.94 (0.17H, m), 5.84 (0.83H, m), 5.69 (1H, m),4.89 (0.13H, m), 4.70 (0.83H, m), 4.25 (1H, m), 4.05 (4H, m), 3.56(0.13H, m), 3.38 (0.87H, m), 2.04 (2.49H, s), 2.03 (0.51H, m), 1.38 (9H,s) ppm (The product was a ˜5:1 mixture of trans- and cis-isomers).

Step 6. [(5S)-5-Amino-5,6-dihydro-2H-pyran-2-yl]methyl acetate

To a solution of{(5S)-5-[(tert-butoxycarbonyl)amino]-5,6-dihydro-2H-pyran-2-yl}methylacetate (205 mg, 0.756 mmol) in methylene chloride (5.2 mL) was added4.0 M hydrogen chloride in dioxane (1.5 mL, 6.0 mmol). The reactionsolution was stirred at room temperature for 6 h. The solvent wasremoved under reduced pressure to give the desired product as whitesolid. LCMS calculated for C₈H₁₄NO₃ (M+H)⁺: m/z=172.1; Found: 172.1.

Step 7.{(5S)-5-[(6-Nitrothieno[3,2-b]pyridin-7-yl)amino]-5,6-dihydro-2H-pyran-2-yl}methylacetate

A mixture of 7-chloro-6-nitrothieno[3,2-b]pyridine (156 mg, 0.727 mmol),[(5S)-5-amino-5,6-dihydro-2H-pyran-2-yl]methyl acetate (129 mg, 0.754mmol) and N,N-diisopropylethylamine (0.26 mL, 1.5 mmol) in isopropylalcohol (1.7 mL) was heated at 90° C. for 2 h. The reaction mixture wasconcentrated and purified with flash chromatography to give the desiredproduct (0.21 g 83%). LCMS calculated for C₁₅H₁₆N₃O₅S (M+H)⁺: m/z=350.1;Found: 350.0.

Step 8.{(5S)-5-[(6-Aminothieno[3,2-b]pyridin-7-yl)amino]tetrahydro-2H-pyran-2-yl}methylacetate

A mixture of{(5S)-5-[(6-nitrothieno[3,2-b]pyridin-7-yl)amino]-5,6-dihydro-2H-pyran-2-yl}methylacetate (210 mg, 0.600 mmol) and 10% palladium on carbon (0.21 g) inmethanol (4.0 mL) was subjected to balloon pressure of H₂ at roomtemperature for 2 h. The mixture was filtered, and the filtrate wasconcentrated and purified with flash chromatography (eluting with 15%methanol in dichloromethane) to give the desired product (145 mg, 75%).LCMS calculated for C₁₅H₂₀N₃O₃S (M+H)⁺: m/z=322.1; Found: 322.0.

Step 9.(1R)-1-{1-[(3S)-6-(Hydroxymethyl)tetrahydro-2H-pyran-3-yl]-H-imidazo[4,5-d]thieno[3,2-b]pyridin-2-yl}ethanol

A mixture of (2R)-2-hydroxypropanamide (131 mg, 1.47 mmol) andtriethyloxonium tetrafluoroborate (263 mg, 1.38 mmol) in THF (2 mL) wasstirred at room temperature for 2 h. The solvent was removed and theresidue dissolved in ethanol (0.85 mL) and added to a suspension of{(5S)-5-[(6-aminothieno[3,2-b]pyridin-7-yl)amino]tetrahydro-2H-pyran-2-yl}methylacetate (145 mg, 0.451 mmol) in ethanol (3.1 mL). The mixture wasstirred at 80° C. for 1 h. The reaction was cooled to room temperatureand diluted with water (1.0 mL). Lithium hydroxide (32.4 mg, 1.35 mmol)was added, and the mixture was stirred for 2 h. The reaction mixture wasdiluted with methanol and purified with prep-LCMS (XBridge C18 column,eluting with a gradient of acetonitrile/water containing 0.1% ammoniumhydroxide, at flow rate of 60 mL/min) to give the desired product aswhite solid (95 mg, 63%). LCMS calculated for C₁₆H₂₀N₃O₃S (M+H)⁺:m/z=334.1; Found: 334.0.

Step 10:((2R,5S)-5-{2-[(1R)-1-Hydroxyethyl]-1H-imidazo[4,5-d]thieno[3,2-b]pyridin-1-yl}tetrahydro-2H-pyran-2-yl)methyl4-methylbenzenesulfonate and((2S,5S)-5-{2-[(1R)-1-hydroxyethyl]-1H-imidazo[4,5-d]thieno[3,2-b]pyridin-1-yl}tetrahydro-2H-pyran-2-yl)methyl4-methylbenzenesulfonate

To a solution of(1R)-1-{1-[(3S)-6-(hydroxymethyl)tetrahydro-2H-pyran-3-yl]-1H-imidazo[4,5-d]thieno[3,2-b]pyridin-2-yl}ethanol(100 mg, 0.300 mmol) (previous step) in methylene chloride (3.4 mL) andpyridine (0.146 mL, 1.80 mmol) was added p-toluenesulfonyl chloride(57.2 mg, 0.300 mmol) and 4-dimethylaminopyridine (1.8 mg, 0.015 mmol)at 0° C. The reaction mixture was allowed to warm to room temperatureovernight. The reaction mixture was concentrated, diluted with methanol,and purified with prep-LCMS (XBridge C18 column, eluting with a gradientof acetonitrile/water containing 0.1% ammonium hydroxide, at flow rateof 60 mL/min) to give two peaks. On analytic HPLC (Waters SunFire C18,2.1×50 mm, 5 μM; Flow rate 3 mL/min; Injection volume 2 μL; At gradientfrom 2 to 80% B in 3 minutes (A=water with 0.025% TFA, B=acetonitrile)):First peak (45.3 mg, 31%) retention time 1.81 min, LCMS calculated forC₂₃H₂₆N₃O₅S₂ (M+H)⁺: m/z=488.1; Found: 488.1. Second peak (8.5 mg, 5.8%)retention time 1.88 min, LCMS calculated for C₂₃H₂₆N₃O₅S₂ (M+H)⁺:m/z=488.1; Found: 488.1.

Step 11.((2R,5S)-5-{2-[(1R)-1-Hydroxyethyl]-1H-imidazo[4,5-d]thieno[3,2-b]pyridin-1-yl}tetrahydro-2H-pyran-2-yl)acetonitrile

A mixture of((2R,5S)-5-{2-[(1R)-1-hydroxyethyl]-1H-imidazo[4,5-d]thieno[3,2-b]pyridin-1-yl}tetrahydro-2H-pyran-2-yl)methyl4-methylbenzenesulfonate (from 1st peak of previous step, 27 mg, 0.055mmol) and sodium cyanide (4.5 mg, 0.092 mmol) in dimethyl sulfoxide (0.4mL) was stirred at 50° C. for 4 h. After cooling, the mixture wasdiluted with methanol and purified with prep-LCMS (XBridge C18 column,eluting with a gradient of acetonitrile/water containing 0.1% ammoniumhydroxide, at flow rate of 30 mL/min) to give the desired product (14.5mg, 76%). LCMS calculated for C₁₇H₁₉N₄O₂S (M+H)⁺: m/z=343.1; Found:343.0. ¹H NMR (DMSO-d₆, 500 MHz) δ 9.51 (1H, s), 8.45 (1H, d, J=5.5 Hz),7.97 (1H, d, J=5.5 Hz), 5.31 (1H, m), 5.20 (1H, m), 4.31 (1H, m), 4.23(1H, m), 4.02 (1H, m), 2.96 (1H, dd, J=17.0 and 4.5 Hz), 2.85 (1H, dd,J=17.0 and 4.5 Hz), 2.66 (1H, m), 2.26 (1H, m), 2.09 (1H, m), 1.73 (1H,m), 1.69 (3H, d, J=6.5 Hz) ppm.

Example 1a.((2R,5S)-5-{2-[(1R)-1-Hydroxyethyl]-1H-imidazo[4,5-d]thieno[3,2-b]pyridin-1-yl}tetrahydro-2H-pyran-2-yl)acetonitrilehydrate

((2R,5S)-5-{2-[(1R)-1-Hydroxyethyl]-1H-imidazo[4,5-d]thieno[3,2-b]pyridin-1-yl}tetrahydro-2H-pyran-2-yl)acetonitrile(52 mg, 0.15 mmol) from Example 25 was crystallized from a mixture ofacetonitrile (8 mL) and water (4 mL). The resulting colorless prismcrystal collected was suitable for X-ray crystal structure analysis.

Crystal data shows: ˜0.520×0.180×0.100 mm, orthorhombic, P212121,a=6.962(3) Å, b=11.531(4) Å, c=20.799(7) Å, Vol=1669.6(10) Å³, Z=4,T=−100° C., Formula weight=359.42, Density=1.430 g/cm³, μ(Mo)=0.22 mm⁻¹.

Data collection was done on a Bruker SMART APEX-II CCD system, MoKalpharadiation, standard focus tube, anode power=50 kV×42 mA, crystal toplate distance=5.0 cm, 512×512 pixels/frame, beam center=(256.13,253.14), total frames=1151, oscillation/frame=0.50°, exposure/frame=10.1sec/frame, SAINT integration, hkl min/max=(−9, 9, −15, 15, −27, 27),data input to shelx=17025, unique data=3975, two-theta range=3.92 to55.72°, completeness to two-theta 55.72=99.80%, R(int-xl)=0.0681, SADABScorrection applied.

Structure was solved using XS(Shelxtl), refined using shelxtl softwarepackage, refinement by full-matrix least squares on F², scatteringfactors from Int. Tab. Vol C Tables 4.2.6.8 and 6.1.1.4, number ofdata=3975, number of restraints=0, number of parameters=235,data/parameter ratio=16.91, goodness-of-fit on F²=1.04, R indices[I>4sigma(I)] R1=0.0505, wR2=0.1242, R indices (all data) R1=0.0769,wR2=0.1401, max difference peak and hole=0.724 and −0.277 e/Å³, refinedflack parameter=−0.12(13), All of the CH hydrogen atoms were refinedusing a riding model. The OH hydrogens were found from a difference mapand fully refined.

Results showed that the asymmetric unit contains one molecule and onewater as shown with thermal ellipsoids drawn to the 50% probabilitylevel. The stereochemistry at each of three stereocenters (as indicatedin the name and structure of the compound above) was confirmed. Theflack parameter refined to 0.28(24) indicating the correct enantiomericsetting.

Example 2.4-[3-(Cyanomethyl)-3-(3′,5′-dimethyl-1H,1′H-4,4′-bipyrazol-1-yl)azetidin-1-yl]-2,5-difluoro-N-[(1S)-2,2,2-trifluoro-1-methylethyl]benzamide

Step 1: 2,4,5-Trifluoro-N-[(1S)-2,2,2-trifluoro-1-methylethyl]benzamide

To a solution of 2,4,5-trifluorobenzoic acid (5.00 g, 28.4 mmol) inacetonitrile (50 mL) was added N,N-dimethylformamide (40 μL) followed byaddition of oxalyl chloride (3.60 mL, 42.6 mmol). After 90 min, thevolatiles were removed under reduced pressure. The residue wasco-evaporated with acetonitrile (50 mL). The residue was then dissolvedin methylene chloride (50 mL). This solution was added drop-wise into acooled (ice bath) mixture of (2S)-1,1,1-trifluoropropan-2-aminehydrochloride (5.52 g, 36.9 mmol) (from Synquest, 98% ee) in toluene(100 mL) and 0.5 M sodium hydroxide aqueous solution (142 mL, 71.0mmol). After addition, the ice bath was removed, and the reaction wasallowed to warm to rt. The reaction was stirred overnight. The organiclayer was separated. The aqueous layer was extracted with methylenechloride (50 mL). The combined organic layers were washed with 20% brine(75 mL) and water (2×75 mL), dried over MgSO₄, filtered and concentratedunder reduced pressure to afford the desired product (6.49 g, 84%) whichwas directly used in the next step without further purification. ¹H NMR(300 MHz, DMSO-d₆) δ 9.01 (d, J=7.6 Hz, 1H), 7.92-7.50 (m, 2H), 4.76 (m,1H), 1.31 (d, J=7.0 Hz, 3H) ppm. LCMS cacld. for C₁₀H₈F₆NO (M+1)⁺:m/z=272.0; Found: 272.0.

Step 2:2,5-Difluoro-4-(3-hydroxyazetidin-1-yl)-N-[(1S)-2,2,2-trifluoro-1-methylethyl]benzamide

A mixture of2,4,5-trifluoro-N-[(1S)-2,2,2-trifluoro-1-methylethyl]benzamide (6.39 g,23.6 mmol), azetidin-3-ol hydrochloride (3.19 g, 28.3 mmol) and1,8-diazabicyclo[5.4.0]undec-7-ene (8.81 mL, 58.9 mmol) in acetonitrile(25 mL) was stirred at 80° C. for 2 h. The reaction mixture was dilutedwith EtOAc (75 mL) and washed with 1N HCl (50 mL), 1N NaHCO₃ (60 mL),20% brine (50 mL) and water (75 mL). The aqueous layers were extractedwith EtOAc (100 mL). The organic layers were combined, dried over MgSO₄,filtered and concentrated under reduced pressure to yield the desiredproduct (7.59 g, 91.8%). ¹H NMR (300 MHz, DMSO-d₆) δ 8.38 (dd, J=8.9,1.9 Hz, 1H), 7.27 (dd, J=12.8, 6.5 Hz, 1H), 6.38 (dd, J=12.3, 7.5 Hz,1H), 5.71 (d, J=6.4 Hz, 1H), 4.74 (dp, J=15.3, 7.6 Hz, 1H), 4.62-4.46(m, 1H), 4.30-4.15 (m, 2H), 3.71 (m, 2H), 1.29 (d, J=7.1 Hz, 3H) ppm.LCMS cacld. for C₁₃H₁₄F₅N₂O₂ (M+1)⁺: m/z=325.1; Found: 325.1.

Step 3:2,5-Difluoro-4-(3-oxoazetidin-1-yl)-N-[(1S)-2,2,2-trifluoro-1-methylethyl]benzamide

To a solution of2,5-difluoro-4-(3-hydroxyazetidin-1-yl)-N-[(1S)-2,2,2-trifluoro-1-methylethyl]benzamide(7.57 g, 23.3 mmol) in methylene chloride (93 mL) was added iodobenzenediacetate (9.40 g, 29.2 mmol) and 2,2,6,6-tetramethyl-1-piperidinyloxyfree radical (1.82 g, 11.7 mmol) (TEMPO) at room temperature. Thereaction mixture was stirred at room temperature overnight. The mixturewas diluted with EtOAc (100 mL), washed with 0.5N NaHCO₃ (2×80 mL), 20%brine (100 mL) and water (100 mL). The aqueous layers were extractedwith ethyl acetate (75 mL). The organic extracts were combined, driedover MgSO₄, filtered and concentrated under reduced pressure. Theresidue was purified by flash chromatography on a silica gel columneluting with 0% to 5% ethyl acetate in methylene chloride to afford thecrude product which was recrystallized from MTBE (50 mL) and heptane(100 mL) to give the desired product (5.44 g, 72%) as colorless solid.¹H NMR (300 MHz, DMSO-d₆) δ 8.52 (d, J=8.0 Hz, 1H), 7.36 (dd, J=12.5,6.5 Hz, 1H), 6.63 (dd, J=12.1, 7.6 Hz, 1H), 4.90 (d, J=2.1 Hz, 4H),4.86-4.68 (m, 1H), 1.31 (d, J=7.1 Hz, 3H) ppm. LCMS cacld. forC₁₃H₁₂F₅N₂O₂ (M+1)⁺: m/z=323.1; Found: 323.0.

Step 4:4-[3-(Cyanomethylene)azetidin-1-yl]-2,5-difluoro-N-[(1S)-2,2,2-trifluoro-1-methylethyl]benzamide

Diethyl cyanomethylphosphonate (1.95 mL, 11.8 mmol) was added drop-wiseto a cooled (ice bath) solution of 1.0 M potassium tert-butoxide in THF(11.8 mL, 11.8 mmol) which was diluted with tetrahydrofuran (12 mL). Thebath was removed and the reaction was warmed to room temperature, andstirred for 90 min. The reaction solution was cooled with an ice bathagain. The above prepared solution was then added over 12 min to acooled (ice-bath) solution of2,5-difluoro-4-(3-oxoazetidin-1-yl)-N-[(1S)-2,2,2-trifluoro-1-methylethyl]benzamide(4.00 g, 12.4 mmol) in tetrahydrofuran (50 mL). The reaction mixture wasstirred for 30 min. The ice bath was removed, and the reaction wasstirred at room temperature overnight, then quenched by the addition of20% brine (75 mL) and ethyl acetate (75 mL). The organic layer wasseparated. The aqueous layer was extracted with ethyl acetate (50 mL).The combined organic layers were dried over MgSO₄, filtered andconcentrated under reduced pressure. The residue was purified by flashchromatography on a silica gel column with ethyl acetate in hexanes (0%to 30%) to yield the desired product (2.6 g). ¹H NMR (400 MHz, DMSO-d₆)δ 8.59-8.37 (m, 1H), 7.33 (dd, J=12.5, 6.4 Hz, 1H), 6.59 (dd, J=12.0,7.4 Hz, 1H), 5.88 (m, 1H), 4.94-4.75 (m, 4H), 4.76 (m, 1H), 1.31 (d,J=7.1 Hz, 3H) ppm. LCMS cacld. for C₁₅H₁₃F₅N₃O (M+1)⁺: m/z=346.1; Found:346.1.

Step 5:4-{3-(Cyanomethyl)-3-[4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazol-1-yl]azetidin-1-yl}-2,5-difluoro-N-[(1S)-2,2,2-trifluoro-1-methylethyl]benzamide

A mixture of 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole(1.00 g, 5.15 mmol),4-[3-(cyanomethylene)azetidin-1-yl]-2,5-difluoro-N-[(1S)-2,2,2-trifluoro-1-methylethyl]benzamide(1.78 g, 5.15 mmol) and 1,8-diazabicyclo[5.4.0]undec-7-ene (0.31 mL, 2.1mmol) in acetonitrile (20.2 mL) was heated at 50° C. overnight. Aftercooling, the solvent was removed under reduced pressure. The residue wasused in the next step without further purification. LCMS cacld. forC₂₄H₂₈BF₅N₅O₃ (M+1)⁺: m/z=540.2; Found: 540.1.

Step 6: 4-[3-(Cyanomethyl)-3-(3′,5′-dimethyl-1H,1′H-4,4′-bipyrazol-1-yl)azetidin-1-yl]-2,5-difluoro-N-[(1S)-2,2,2-trifluoro-1-methylethyl]benzamide

A mixture of4-{3-(cyanomethyl)-3-[4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazol-1-yl]azetidin-1-yl}-2,5-difluoro-N-[(1S)-2,2,2-trifluoro-1-methylethyl]benzamide(329 mg, 0.610 mmol), 4-bromo-3,5-dimethyl-1H-pyrazole (206 mg, 1.18mmol), tetrakis(triphenylphosphine)palladium(0) (110 mg, 0.098 mmol) andsodium carbonate (320 mg, 3.0 mmol) in 1,4-dioxane (10 mL)/water (5 mL)was purged with nitrogen and stirred at 110° C. for 1 h. The reactionmixture was diluted with EtOAc, washed with water and brine,concentrated. The residue was purified first with silica gel (elutingwith 0-100% EtOAc/hexanes followed by 10% methanol/dichloromethane), andthen by prep-LCMS (XBridge C18 column, eluting with a gradient ofacetonitrile/water containing 0.1% ammonium hydroxide, at flow rate of60 mL/min) to give the desired product (30 mg, 9.7%). ¹H NMR (500 MHz,DMSO-d₆) δ 12.17 (1H, s), 8.45 (1H, d, J=8.0 Hz), 8.10 (1H, s), 7.70(1H, s), 7.34 (1H, m), 6.61 (1H, s), 4.77 (1H, m), 4.62 (2H, d, J=9.0Hz), 4.39 (1H, d, J=9.0 Hz), 3.64 (2H, s), 2.22 (6H, s), 1.31 (6H, d,J=7.0 Hz) ppm. LCMS calculated for C₂₃H₂₃F₅N₇O (M+H)⁺: m/z=508.2; Found:508.0.

Example A: In Vitro JAK Kinase Assay

Compounds herein were tested for inhibitory activity of JAK targetsaccording to the following in vitro assay described in Park et al.,Analytical Biochemistry 1999, 269, 94-104. The catalytic domains ofhuman JAK1 (a.a. 837-1142), JAK2 (a.a. 828-1132) and JAK3 (a.a.781-1124) were expressed using baculovirus in insect cells and purified.The catalytic activity of JAK1, JAK2 or JAK3 was assayed by measuringthe phosphorylation of a biotinylated peptide. The phosphorylatedpeptide was detected by homogenous time resolved fluorescence (HTRF).IC₅₀s of compounds were measured for each kinase in the 40 μL reactionsthat contain the enzyme, ATP and 500 nM peptide in 50 mM Tris (pH 7.8)buffer with 100 mM NaCl, 5 mM DTT, and 0.1 mg/mL (0.01%) BSA. For the 1mM IC₅₀ measurements, ATP concentration in the reactions was 1 mM.Reactions were carried out at room temperature for 1 hour and thenstopped with 20 μL 45 mM EDTA, 300 nM SA-APC, 6 nM Eu-Py20 in assaybuffer (Perkin Elmer, Boston, Mass.). Binding to the Europium labeledantibody took place for 40 minutes and HTRF signal was measured on aPHERA star plate reader (BMG, Cary, N.C.). The data for the JAK1 and/orJAK2 inhibitors were obtained by testing the compounds in the Example Aassay at 1 mM ATP.

Example B: PI3Kδ Scintillation Proximity Assay

Materials

[γ-³³P]ATP (10 mCi/mL) was purchased from Perking Elmer (Waltham,Mass.). Lipid kinase substrate, D-myo-Phosphatidylinositol4,5-bisphosphate (PtdIns(4,5)P2)D (+)-sn-1,2-di-O-octanoylglyceryl,3-O-phospho linked (PIP2), CAS 204858-53-7, was purchased from EchelonBiosciences (Salt Lake City, Utah). PI3Kδ (p110δ/p85α) was purchasedfrom Millipore (Bedford, Mass.). ATP, MgCl₂, DTT, EDTA, MOPS and CHAPSwere purchased from Sigma-Aldrich (St. Louis, Mo.). Wheat GermAgglutinin (WGA) YSi SPA Scintillation Beads was purchased from GEhealthcare life sciences (Piscataway, N.J.).

The kinase reaction was conducted in polystyrene 384-well matrix whiteplate from Thermo Fisher Scientific in a final volume of 25 μL.Inhibitors were first diluted serially in DMSO and added to the platewells before the addition of other reaction components. The finalconcentration of DMSO in the assay was 0.5%. The PI3K assays werecarried out at room temperature in 20 mM MOPS, pH 6.7, 10 mM MgCl₂, 5 mMDTT and CHAPS 0.03%. Reactions were initiated by the addition of ATP,the final reaction mixture consisted of 20 μM PIP2, 20 μM ATP, 0.2 μCi[γ-³³P] ATP, 4 nM PI3Kδ. Reactions were incubated for 210 min andterminated by the addition of 40 μL SPA beads suspended in quenchbuffer: 150 mM potassium phosphate pH 8.0, 20% glycerol. 25 mM EDTA, 400μM ATP. The final concentration of SPA beads was 1.0 mg/mL. After theplate sealing, plates were shaken overnight at room temperature andcentrifuged at 1800 rpm for 10 minutes, the radioactivity of the productwas determined by scintillation counting on Topcount (Perking Elmer).IC₅₀ determination was performed by fitting the curve of percent controlactivity versus the log of the inhibitor concentration using theGraphPad Prism 3.0 software. The data for the PI3Kδ inhibitors wereobtained by testing the compounds in the Example B assay.

Example C: Pfeiffer Model of Lymphoma

Methods:

Female SCID mice, (5 to 8 weeks of age, Charles River Laboratories,Wilmington, Mass.) were inoculated with 1×107 tumor cells (Pfeiffer,ATCC #CRL-2632, Manassas, Va.) and matrigel (BD Biosciences #354234) in0.2 mL sterile saline. The inoculation was performed subcutaneously onthe flank. Tumor tissue fragments (approximately 3 mm×3 mm) werecollected 3 to 6 weeks after the inoculation of cultured cells andimplanted subcutaneously in lieu of cellular inoculation. Tissuefragments were implanted as solid pieces using blunt-tip forceps. Thetreatment of tumor bearing mice was started 15 to 25 days after tumorinoculation, depending upon the tumor size. Animals were sorted toobtain roughly equivalent mean tumor volumes in each group. Minimum meantumor volume in all groups was 150 mm3 on the first day of treatment andgroups consisted of 7 animals. Experimental therapeutic agent, Example347, was administered to mice orally (PO). Treatment frequency was 2times daily for a minimum of 14 days for efficacy. The size ofsubcutaneous tumors was measured 2 to 3 times weekly using a digitalcaliper. The tumor volume was calculated by measuring the tumor in 2dimensions and utilizing the equation: Volume=[Length×(Width2)]/2; wherethe larger number was length, and the smaller number width. If multipletumors were formed, the final volume was the sum of the individualtumors subject to the same equation: eg, 2 tumors;Volume={[L1×(W1)2]/2}+{[L2×(W2)2]/2}. Effects on tumor growth werereported as percent tumor growth inhibition (% TGI). Percent TGI wascalculated with the equation: (1−(Tx vol./control vol.))*100, wherecontrol volume was the vehicle or untreated tumor volume on a given day,and Tx volume was any treatment group tumor volume on that same day.Statistical differences between treatment and vehicle controls wereassessed using ANOVA: Single Factor test.

Results:

Compound 32c (Table 2 supra) was evaluated as a single agent in thePfeiffer human tumor xenograft model of diffuse large B-cell lymphoma, asubtype of NHL. Pfeiffer cancer cells were shown to be sensitive to theanti-proliferative effects of Example 347 in vitro. Therefore, a tumormodel was established based on subcutaneous inoculation of tumor cellsinto immune compromised SCID mice and tumor-bearing mice received twicedaily oral doses of vehicle or Compound 32c at 0.3, 1, 3, or 10 mg/kgfor 14 days. Compound 32c treatment inhibited tumor growth by 22%, 24%,36%, and 58% (percent tumor growth inhibition) with increasing dose.

Example D. Western-Blot Analysis

The following materials and methods were used in the Western blotanalysis infra. Cells (5 million) were lysed in a 300 μl volume of lysisbuffer. Soluble fractions were collected by centrifugation. 25 μl ofcell lysate were loaded into Tris-Glycine polyacrylamide gels andsubjected to electrophoresis. The proteins were transferred intonitrocellulose membrane and probed with antibodies from Cell SignalingTechnology for the following proteins: phospho-Stat3 Y705, phospho-Akt5473, pim1, pim2, pim3, c-myc, phospho-p70S6K, phosho-S6, phospho-BadS112 and actin.

Example E. Levels of IL6 and IL10 in Cell Lines

High levels of IL6 and IL10 were seen in various DLBCL cell lines (FIG.1A). IL6 and IL10 were also shown to activate JAK/STAT signaling withIL10 being a stronger activator of JAK/STAT signaling than IL6 across apanel of DLBCL cell lines (FIG. 1B). High levels of IL6 and IL10 arepresent in serum from DLBCL patients and correlate with shorter eventfree survival and a higher International Prognostic Index score.

Example F. IL10 Makes Pfeiffer Cells Resistant to PI3Kδ Inhibition andcan be Reversed by JAK1/2 or JAK1 Blockade

Cell Proliferation Assay

Diffuse large B cell lymphoma cells were seeded at 2000 cell/well in 96well culture plates in the absence or presence of 10 ng/ml IL10.Compounds were added to these cells after dilution in DMSO first,followed by dilution in culture medium (4× concentration). The cellswere cultured in an incubator for 3 days with 5% CO₂. Cell proliferationwas assessed using the Cell titer-glow assay (Promega, Madison, Wis.).The cell proliferation assay was carried out first in Pfeiffer cells(germinal center B cell (GCB) diffuse large B cell lymphoma (GCB-DLBCL)cells) and HBL-1 cells (activated B-cell like (ABC) diffuse large B celllymphoma (ABC-DLBCL)).

FIG. 2A depicts % inhibition in the cellular proliferation assay inPfeiffer cells as a function of the concentration of Compound 28 (aPI3Kδ inhibitor) with vehicle (DMSO), DMSO+IL10, andDMSO+IL10+ruxolitinib (a JAK1/JAK2 inhibitor). FIG. 2B depicts %inhibition in the cellular proliferation assay in Pfeiffer cells as afunction of the concentration of Compound 28 (a PI3Kδ inhibitor) withvehicle (DMSO), DMSO+IL10, and DMSO+IL10+Compound 7 (a selective JAK1inhibitor). The results show that IL10 makes Pfeiffer cells resistant toPI3Kδ inhibition, but that this resistance can be reversed by blockingJAK1 and/or JAK2 signaling. Hence, a synergistic effect on Pfeiffer cellproliferation when a PI3Kδ inhibitor and a JAK1 and/JAK2 inhibitor areused in combination. Synergy was also observed without the IL10.Induction of apoptosis was also observed with this combination.

Similar results were observed in HBL-1 cells with ruxolitinib.Accordingly, FIG. 3 depicts % inhibition in the cellular proliferationassay in HBL-1 cells as a function of the concentration of Compound 28(a PI3Kδ inhibitor) with vehicle (DMSO), DMSO+IL10, andDMSO+IL10+ruxolitinib (a JAK1/JAK2 inhibitor).

Example G. IL10-Induced Expression of Pim2 is Blocked by a JAK1/2Inhibitor

Pfeiffer cells were treated for 24 hours with vehicle (DMSO),ruxolitinib (18424), Compound 28, or Compound 28 and ruxolitinib (18424)with or without IL10 and then subjected to Western blot analysis toprobe for the following proteins: phospho-Stat3 Y705, phospho-Akt S473,Pim2, c-Myc, phospho-p70S6K, phosho-S6, phospho-Bad S112 and actin. FIG.4 shows that IL10-induced expression of Pim2 is blocked by ruxolitinib(a JAK1/JAK2 inhibitor). IL6 and IL10 promote cell survival through theexpression of Pim2, which is dependent on JAK1 activity. FIG. 4 alsoshows a synergistic reduction of c-Myc and P-S6 in the presence ofcombined Compound 28 and ruxolitinib treatment. The reduction of c-Mycprotein may be responsible for the synergistic effect for thecombination treatment.

Example H. IL10-Induced Expression of Pim2 is Blocked by a SelectiveJAK1 Inhibitor

Pfeiffer cells were treated for 24 hours with vehicle (DMSO), Compound7, Compound 28, or Compound 28 and Compound 7 with or without IL10 andthen subjected to Western blot analysis to probe for the followingproteins: phospho-Stat3 Y705, phospho-Akt S473, Pim2, c-Myc,phospho-p70S6K, phosho-S6, phospho-Bad S112 and actin. FIG. 5 shows thatIL10-induced expression of Pim2 is blocked by a selective JAK1 inhibitor(Compound 7).

Example I. Increase in Potency of PI3Kδ Inhibitor by Selective JAK1Inhibitor

To test the effects of IL-10 on cell growth and sensitivity to BCRpathway inhibition, the Pfeiffer cell line was used as a model system ofDLBCL. Pfeiffer cells are of the germinal center B-cell (GCB) subtype ofDLBCL, have been shown to express PI3Kδ, are sensitive to PI3Kδinhibition, and activate the JAK/STAT pathway in response to multiplecytokines as shown above. Pfeiffer cells were treated for 3 days withvarious concentrations of Compound 28 in the presence or absence ofIL-10 and 1 μM of Compound 16, and cell growth was measured using an ATPreadout (see table below). As shown in FIG. 6, the presence of IL-10shifted the potency of Compound 28 by ˜10-fold (IC₅₀=0.67 μM, −IL-10;IC₅₀=6.36 μM, +IL-10). Addition of the JAK1 inhibitor, Compound 16,reversed this effect so that the combination was ˜50-fold more potent.In this system, the JAK1 inhibitor alone had no effect (IC₅₀>1 μM).Further, as shown in FIG. 7 (showing Annexin-V staining of Pfeiffercells treated for 3 days in 10% FBS+IL10; Compound 16 was tested at 1μM), inhibition of PI3Kδ together with JAK1 signaling led to increasedapoptosis whereas neither agent alone had a significant effect.

10% FBS + IL10 Compound 28 + Compound 28 Compound 16 Compound 16 IC₅₀(μM) 6.36 >1 0.035

Example J. Effect of Combined JAK1 and PI3Kδ Treatment on STAT3Phosphorylation and pAKT Inhibition

To assess the effects on downstream signaling pathways, Pfeiffer cellswere treated with Compound 28+/−Compound 16 for 4 hours and thenstimulated with IL-10 for 15 minutes. Extracts were analyzed by Westernblot for pAKT and pSTAT3. As shown in FIG. 8, the AKT pathway wasconstitutively activated in Pfeiffer cells. Blockade of PI3Kδ signalingled to complete inhibition of pAKT whereas treatment with the JAK1inhibitor had no effect. In contrast, Compound 16 led to inhibition ofSTAT3 phosphorylation whereas the PI3Kδ inhibitor did not. Thecombination of both compounds was required to block both pathways.

All patents, patent publications, and journal articles referred to supraare incorporated herein by reference in their entireties.

What is claimed is:
 1. A method of treating myelofibrosis in a patientin need thereof, comprising administering to the patient (a) aninhibitor of JAK1/2, which is(3R)-3-cyclopentyl-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]propanenitrile,or a pharmaceutically acceptable salt thereof, and (b) an inhibitor ofPI3Kδ, which is4-{3-[1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-5-chloro-2-ethoxy-6-fluorophenyl}pyrrolidin-2-one,and enantiomers and diastereomers thereof, or a pharmaceuticallyacceptable salt thereof.
 2. The method of claim 1, wherein the inhibitorof JAK1/2 is(3R)-3-cyclopentyl-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]propanenitrilephosphoric acid salt.
 3. The method of claim 1, wherein the inhibitor ofPI3Kδ is(S)-4-(3-((S)-1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-5-chloro-2-ethoxy-6-fluorophenyl)pyrrolidin-2-one,or a pharmaceutically acceptable salt thereof.
 4. The method of claim 1,wherein the inhibitor of PI3Kδ is(R)-4-(3-((S)-1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-5-chloro-2-ethoxy-6-fluorophenyl)pyrrolidin-2-one,or a pharmaceutically acceptable salt thereof.
 5. The method of claim 1,wherein the inhibitor of PI3Kδ is(S)-4-(3-((R)-1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-5-chloro-2-ethoxy-6-fluorophenyl)pyrrolidin-2-one,or a pharmaceutically acceptable salt thereof.
 6. The method of claim 1,wherein the inhibitor of PI3Kδ is(R)-4-(3-((R)-1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-5-chloro-2-ethoxy-6-fluorophenyl)pyrrolidin-2-one,or a pharmaceutically acceptable salt thereof.
 7. The method of claim 1,wherein the inhibitor of JAK1/2 and the inhibitor of PI3Kδ areadministered simultaneously.
 8. The method of claim 1, wherein theinhibitor of JAK1/2 and the inhibitor of PI3Kδ are administeredsequentially.
 9. A method of treating myelofibrosis in a patient in needthereof, comprising administering to the patient: (a) an inhibitor ofJAK1/2, which is(3R)-3-cyclopentyl-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]propanenitrilephosphoric acid salt; and (b) an inhibitor of PI3Kδ, which is selectedfrom:(S)-4-(3-((S)-1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-5-chloro-2-ethoxy-6-fluorophenyl)pyrrolidin-2-one;(R)-4-(3-((S)-1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-5-chloro-2-ethoxy-6-fluorophenyl)pyrrolidin-2-one;(S)-4-(3-((R)-1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-5-chloro-2-ethoxy-6-fluorophenyl)pyrrolidin-2-one;and(R)-4-(3-((R)-1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-5-chloro-2-ethoxy-6-fluorophenyl)pyrrolidin-2-one;and pharmaceutically acceptable salts of any of the aforementioned. 10.The method of claim 9, wherein the inhibitor of PI3Kδ is(S)-4-(3-((S)-1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-5-chloro-2-ethoxy-6-fluorophenyl)pyrrolidin-2-one,or a pharmaceutically acceptable salt thereof.
 11. The method of claim9, wherein the inhibitor of PI3Kδ is(R)-4-(3-((S)-1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-5-chloro-2-ethoxy-6-fluorophenyl)pyrrolidin-2-one,or a pharmaceutically acceptable salt thereof.
 12. The method of claim9, wherein the inhibitor of PI3Kδ is(S)-4-(3-((R)-1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-5-chloro-2-ethoxy-6-fluorophenyl)pyrrolidin-2-one,or a pharmaceutically acceptable salt thereof.
 13. The method of claim9, wherein the inhibitor of PI3Kδ is(R)-4-(3-((R)-1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-5-chloro-2-ethoxy-6-fluorophenyl)pyrrolidin-2-one,or a pharmaceutically acceptable salt thereof.
 14. The method of claim9, wherein the inhibitor of JAK1/2 and the inhibitor of PI3Kδ areadministered simultaneously.
 15. The method of claim 9, wherein theinhibitor of JAK1/2 and the inhibitor of inhibitor of PI3Kδ areadministered sequentially.